BACKGROUND
archival formalin-fixed, paraffin-embedded  tissue samples represent a robust and invaluable source of human tissue for gene expression analysis.
compared to fresh and snap frozen tissue, ffpe tissue has an inherent advantage in that retrospective patient data, including survival history and treatment response etc, is readily available, allowing immediate comparison with clinical pathological parameters.
data generated can potentially highlight biomarkers useful in disease classification, diagnosis and prognosis, and potentially elucidate novel therapeutic targets  <cit> .
however, these tissues have not been widely used in molecular biology due to the degradation and chemical modification of rna extracted from ffpe blocks.
rna extracted from ffpe is degraded to fewer than  <dig> bases  <cit>  in length because archived blocks are often stored at room temperature for long periods of time.
the situation is made more complicated by the fact that rna is modified by methylol groups to form cross-links with protein or nucleic acid during formalin fixation  <cit> , which results in poor yields  <cit>  and compromised extracts.
real-time quantitative taqman® reverse transcriptase-polymerase chain reaction  analysis has been introduced as a sensitive, accurate, and highly reproducible method to study gene expression  <cit> .
it has been successfully used to detect gene transcript levels from snap frozen tissue extracts and even from ffpe containing partially fragmented rna  <cit>  although the detection rate is lower as indicated for example by invariably higher ct values in the latter  <cit> .
in this study we examined and optimized selected rna extraction protocols, including stratagene absolutely rna® ffpe kit and ambion recoverall™ total nucleic acid isolation kit, by comparison of parallel extracts from ffpe and snap frozen cell preparations using a cell line model .
we further analyzed these extracts using different taqman® protocols, including two types of taqman® master mix  and gene expression master mix ) and newly developed taqman® with pre-amplification method , with a panel of assays over a range of amplicon sizes.
RESULTS
rna extraction
rna quantity was assessed spectrophotometrically using nanodrop® nd- <dig> spectrophotometer , which showed that the yields from snap frozen extracts were greater than those from ffpe when rna was extracted from identical numbers of cells using all the protocols examined .
in comparison of ffpe extracts , ambion gave the highest yields, and column based stratagene and ambion protocols produced clean rna with od 260/ <dig> ratio greater than  <dig> .
rna quality was assessed using agilent  <dig> bioanalyzer  and taqman® rt-pcr, which showed variations in rna quality dependant on the protocol used.
stratagene and ambion recoverall™ kits gave superior ffpe rna results with regard to quality than the others examined.
evaluation of modified protocols
modification to the stratagene and ambion protocols generated approximately 25–40% greater yields and larger fragments of rna  than the standard procedure.
adjustment to stratagene and ambion protocols produced decreased cts , indicating the improved quality of rna extracted from ffpe .
a separate experiment using ambion recoverall™ kit enabled extraction of large rna molecules including cross-linked rnas .
incubation of eluted rna at 70°c for  <dig> minutes was found to be the best condition to disrupt cross-links while not compromising rna integrity in comparison with other modifications .
comparison of taqman® universal pcr master mix , gene expression master mix  and preamp
ct values were also dependent on the type of taqman® pcr master mix that was used .
taqman® with upmm generated higher cts for long amplicons than gemm.
this was observed by a comparison of two gapdh assays – one  <dig> bp and the other  <dig> bp.
there was a mean threshold detection difference of  <dig> cycles using upmm and a mean difference of  <dig> cycles using gemm with rna template extracted using ambion recoverall™ kit .
in addition, hla_a is the largest amplicon  which produced no product with upmm, however, gemm and preamp both allowed the detection of this amplicon size .
gemm improved the ct values for long amplicons  compared with upmm .
when the Δcts were compared to theoretical Δcts, preamp results generally correlated with gemm for all the  <dig> assays analyzed .
evaluation of taqman® preamp using cdkn1b
taqman® preamp analysis consistently achieved decreased ct values in both snap frozen and ffpe aliquots compared with taqman® without pre-amplification step .
a good correlation between preamp and upmm was observed using cdkn1b to analyze rna extracted with different protocols , suggesting that preamp does not introduce any bias into the reaction.
comparison of ct difference between ffpe and snap frozen cells
taqman® analysis over all assays showed cts to be  <dig> to  <dig> cycles higher when using ffpe extracts compared to snap frozen counterparts when the amounts of input rna were identical .
focussing on an analysis of gapdh using two assays with different amplicon sizes revealed the smaller amplicon assay  produced cts from snap frozen and ffpe samples that were closer together .
discussion
rna analysis has generally been performed on snap-frozen or fresh materials, using variable techniques including microarray, northern blotting and rt-pcr.
however, a constant challenge has been robust and reliable analysis of gene expression in archival tissues.
this application has been frustrated by poor rna yields, small sizes of extracted fragments and low levels of detectable rna in the extracts  <cit> .
in this study we established a cell line model to test modified extraction protocols and quantify differences in performance using a panel of qrt-pcr assays on ffpe compared with parallel snap-frozen cell preparations using taqman® methodology.
we found that our modified extraction method and taqman® preamp enhanced expression analysis from ffpe cells optimally.
rna and ffpe
generally, rna extracted from ffpe materials is fragmented and chemically modified.
fragmentation occurs possibly since tissues are surgically removed, and it continuously occurs during fixation and preservation.
experimental design was such that equal numbers of cells were processed for ffpe cell block construction and snap freezing for comparative purposes.
by using cells fixed under controlled conditions possible variables due to the effects of storage on rna degradation were negated, thus revealing the true impact of formalin fixation on rna quality.
we extracted intact rna fragments in a separate experiment from pellets with a large number of formalin fixed paraffin embedded normal thyroid cells , which was consistent with a finding by scicchitana et al  <cit> .
in that study, intact rna molecules were extracted from newly formalin fixed paraffin embedded human bone marrow stromal cells suggesting that rna degradation could be a minor problem for recently fixed ffpe in some types of cells.
our study showed that the differences in rna quality between the small and large fixed pellets  might be due to the fact that the majority of cells in the centre of the large pellet were remained alive during construction of the large solid cell pellet.
this could limit the access of rnases to intracellular mrna because degradation will not fully occur until the fixation process begins resulting to the intact mrna being maintained.
unfortunately, intact mrna is not generally obtained in majority of fixed tissues or cell lines, and rna extracted from ffpe is normally degraded to fewer than  <dig> bp.
extraction protocols
a prerequisite for gene expression studies in formalin-fixed tissue samples was the establishment of a reliable and reproducible extraction method to provide detectable rnas for subsequent analysis  <cit> .
the quality of extracted rna can be variable among different extraction methods due to several factors, such as the contamination of rnases, proteins and genomic dna  <cit> .
our results showed that proteinase k digestion based protocols followed by on column dnase digestion and rna elution produced good detectable rna of ffpe.
proteinase k digestion has also been demonstrated by many laboratories to be critical in rna extraction protocol  <cit>  possibly because of two functions.
on one hand, it can degrade proteins that are covalently cross-linked with each other and nucleic acid to release rna from the matrix, thereby allowing efficient rna extraction from ffpe materials  <cit> .
on the other, it can inactivate rnases that tend to be stable and do not require cofactors to function.
thus its activity avoids any potential reactivation of rnase during reversal of fixation in aqueous buffers  <cit> .
our data demonstrates that incubation in proteinase k buffer at 70°c for  <dig> minutes facilitated the disruption of cross-links, resulting in improved quantity and quality of rna.
the rna extracted using the modified protocols  enhanced detection by a mean of  <dig> cycles , which is equivalent to  <dig> fold increased sensitivity.
it is most likely that the additional incubation step removed the remaining cross-links between rna and protein leading to the longer rna molecules being extracted  <cit> .
in addition, this incubation denatured proteinase k, thus avoided its further damage to rna in the following purification procedure, which suggests the necessity of a heating  application in any proteinase k based extraction of ffpe samples.
taqman® preamp
taqman® qrt-pcr technique is based on the 5' nuclease activity of taq dna polymerase and involves cleavage of a specific fluorogenic hybridization probe that is flanked by pcr primers  <cit> .
because of the small target size, many laboratories have demonstrated that it is possible to measure gene expression levels using ffpe tissues as a source of mrna  <cit> .
still, it seems to be problematic to perform large scale of analysis on ffpe because of the high cts and limited concentration of extracts  <cit> .
we employed a novel taqman® preamp technique which we found to be a practical solution to decrease ct values, and in particular suitable in our hands to generate real-time pcr results from limited amounts of input rna, such as extracted from laser captured microdissected material  <cit> .
the principle of taqman® preamp technique is to amplify target cdna prior to real-time taqman® pcr analysis.
briefly, cdna is synthesized from total rna by use of random priming.
the cdna for the specific target assays is then amplified by pre-amplification reaction using pooled gene-specific primers to increase the number of targeted copies.
the pre-amplification product is diluted and finally analyzed by real-time taqman® pcr using single assay containing one pair of gene-specific primers and probe.
the taqman® preamp technique addresses the challenge faced by researchers working with rare or precious samples, which only limited rna could be extracted from, to perform gene expression analyses using real-time qrt-pcr.
the simple process enables the user to perform uniform amplification from as little as one nanogram of cdna  or alternatively conduct up to  <dig> real-time pcr reactions per pre-amplification reaction without compromising the available sample material.
our results demonstrated that taqman® preamp overcame the difficulties usually caused by low yields of rna extraction from ffpe.
influence of taqman® master mix and amplicon length
this study demonstrated that the sensitivity of taqman® detection was influenced by choice of master mix and amplicon length in assay design.
we evaluated upmm and gemm which showed similar sensitivity for short amplicons , while gemm displayed better sensitivity for longer amplicons  compared with upmm in parallel snap frozen and ffpe extracts .
this effect was dissipated when upmm was used after pre-amplification, possibly because of the increased copy number of template available or because of the composition of the preamp mastermix which is closer in components to gemm than upmm.
reagents can have a significant effect on assay reproducibility  <cit>  due to some parameters such as different polymerases sensitivity  <cit> , primer binding efficiency and the concentration of mg2+  <cit> .
karrer et al.
described the monte carlo effect using plant material suggesting that the pcr reproducibility could be limited when the number of available templates is low.
increased concentrations of mg2+ reduced pcr variation possibly by allowing a higher proportion of annealed primers extended by the more active polymerase  <cit> .
our data corroborated the observation that amplicon size is crucial in designing assays to analyze gene expression levels not only using ffpe extracts but also using rnas with high integrity  <cit> .
many researchers have found that short amplicons generated lower cts than longer amplicons on analysis of the same gene in ffpe  <cit> .
data generated in this study evaluating snap frozen samples using two sizes of gapdh  demonstrated gapdh- <dig> generated a reduction in ct by  <dig> –  <dig> cycles over gapdh- <dig> in both gemm and preamp experiments, demonstrating that smaller amplicons give more consistent results  <cit> .
in addition, we found cts between ffpe and snap frozen were closer for small amplicons than that for large amplicons .
for example, the analysis of gapdh using a target amplicon of  <dig> bp displayed cts  <dig> –  <dig>  cycles closer between ffpe and snap frozen than the same experiment using the longer amplicon size of  <dig> bp.
we further measured the amplification efficiency associated with these two sizes of gapdh assays using a broad dilution range  which showed closed efficiencies with  <dig> % for gapdh- <dig> and  <dig> % for gapdh- <dig> .
this measurement eliminates non-specific amplification that could contribute to decreased amplification efficiency of the true target.
therefore, it seems reasonable to conclude that the shift of efficiency detected from frozen to fixed material  is due to degradation of rna in ffpe, and logical to extrapolate that the longer an amplicon is, the more likely its template will be degraded in extracted rna.
as a general rule, house-keeping genes or normalising assays should have amplicon sizes that match the size of the target whose expression is to be measured  <cit>  and amplicons less than  <dig> bp should be employed in gene expression studies using ffpe materials.
CONCLUSIONS
we evaluated the effect of modifying recommended extraction protocols to reproducibly produce rna that may be successfully amplified using qrt-pcr.
we have found the taqman® preamp system to be a robust and practical solution to limited quantities of rna and have demonstrated comparable results in matched ffpe and snap frozen preparations providing proof of principle that this method may reliably be utilised in the context of multiple expression analyses from individual ffpe samples.

 archival formalin-fixed, paraffin-embedded tissue samples represent a robust and invaluable source of human tissue for gene expression analysis. compared to fresh and snap frozen tissue, ffpe tissue has an inherent advantage in that retrospective patient data, including survival history and treatment response etc, is readily available, allowing immediate comparison with clinical pathological parameters. data generated can potentially highlight biomarkers useful in disease classification, diagnosis and prognosis, and potentially elucidate novel therapeutic targets. however, these tissues have not been widely used in molecular biology due to the degradation and chemical modification of rna extracted from ffpe blocks. rna extracted from ffpe is degraded to fewer than bases in length because archived blocks are often stored at room temperature for long periods of time. the situation is made more complicated by the fact that rna is modified by methylol groups to form cross-links with protein or nucleic acid during formalin fixation, which results in poor yields and compromised extracts. real-time quantitative taqman® reverse transcriptase-polymerase chain reaction analysis has been introduced as a sensitive, accurate, and highly reproducible method to study gene expression. it has been successfully used to detect gene transcript levels from snap frozen tissue extracts and even from ffpe containing partially fragmented rna although the detection rate is lower as indicated for example by invariably higher ct values in the latter. in this study we examined and optimized selected rna extraction protocols, including stratagene absolutely rna® ffpe kit and ambion recoverall™ total nucleic acid isolation kit, by comparison of parallel extracts from ffpe and snap frozen cell preparations using a cell line model. we further analyzed these extracts using different taqman® protocols, including two types of taqman® master mix and gene expression master mix ) and newly developed taqman® with pre-amplification method, with a panel of assays over a range of amplicon sizes.  rna extraction rna quantity was assessed spectrophotometrically using nanodrop® nd- spectrophotometer, which showed that the yields from snap frozen extracts were greater than those from ffpe when rna was extracted from identical numbers of cells using all the protocols examined. in comparison of ffpe extracts, ambion gave the highest yields, and column based stratagene and ambion protocols produced clean rna with od 260/ ratio greater than. rna quality was assessed using agilent bioanalyzer and taqman® rt-pcr, which showed variations in rna quality dependant on the protocol used. stratagene and ambion recoverall™ kits gave superior ffpe rna results with regard to quality than the others examined. evaluation of modified protocols modification to the stratagene and ambion protocols generated approximately 25–40% greater yields and larger fragments of rna than the standard procedure. adjustment to stratagene and ambion protocols produced decreased cts, indicating the improved quality of rna extracted from ffpe. a separate experiment using ambion recoverall™ kit enabled extraction of large rna molecules including cross-linked rnas. incubation of eluted rna at 70°c for minutes was found to be the best condition to disrupt cross-links while not compromising rna integrity in comparison with other modifications. comparison of taqman® universal pcr master mix, gene expression master mix and preamp ct values were also dependent on the type of taqman® pcr master mix that was used. taqman® with upmm generated higher cts for long amplicons than gemm. this was observed by a comparison of two gapdh assays – one bp and the other bp. there was a mean threshold detection difference of cycles using upmm and a mean difference of cycles using gemm with rna template extracted using ambion recoverall™ kit. in addition, hla_a is the largest amplicon which produced no product with upmm, however, gemm and preamp both allowed the detection of this amplicon size. gemm improved the ct values for long amplicons compared with upmm. when the Δcts were compared to theoretical Δcts, preamp results generally correlated with gemm for all the assays analyzed. evaluation of taqman® preamp using cdkn1b taqman® preamp analysis consistently achieved decreased ct values in both snap frozen and ffpe aliquots compared with taqman® without pre-amplification step. a good correlation between preamp and upmm was observed using cdkn1b to analyze rna extracted with different protocols, suggesting that preamp does not introduce any bias into the reaction. comparison of ct difference between ffpe and snap frozen cells taqman® analysis over all assays showed cts to be to cycles higher when using ffpe extracts compared to snap frozen counterparts when the amounts of input rna were identical. focussing on an analysis of gapdh using two assays with different amplicon sizes revealed the smaller amplicon assay produced cts from snap frozen and ffpe samples that were closer together. discussion rna analysis has generally been performed on snap-frozen or fresh materials, using variable techniques including microarray, northern blotting and rt-pcr. however, a constant challenge has been robust and reliable analysis of gene expression in archival tissues. this application has been frustrated by poor rna yields, small sizes of extracted fragments and low levels of detectable rna in the extracts. in this study we established a cell line model to test modified extraction protocols and quantify differences in performance using a panel of qrt-pcr assays on ffpe compared with parallel snap-frozen cell preparations using taqman® methodology. we found that our modified extraction method and taqman® preamp enhanced expression analysis from ffpe cells optimally. rna and ffpe generally, rna extracted from ffpe materials is fragmented and chemically modified. fragmentation occurs possibly since tissues are surgically removed, and it continuously occurs during fixation and preservation. experimental design was such that equal numbers of cells were processed for ffpe cell block construction and snap freezing for comparative purposes. by using cells fixed under controlled conditions possible variables due to the effects of storage on rna degradation were negated, thus revealing the true impact of formalin fixation on rna quality. we extracted intact rna fragments in a separate experiment from pellets with a large number of formalin fixed paraffin embedded normal thyroid cells, which was consistent with a finding by scicchitana et al. in that study, intact rna molecules were extracted from newly formalin fixed paraffin embedded human bone marrow stromal cells suggesting that rna degradation could be a minor problem for recently fixed ffpe in some types of cells. our study showed that the differences in rna quality between the small and large fixed pellets might be due to the fact that the majority of cells in the centre of the large pellet were remained alive during construction of the large solid cell pellet. this could limit the access of rnases to intracellular mrna because degradation will not fully occur until the fixation process begins resulting to the intact mrna being maintained. unfortunately, intact mrna is not generally obtained in majority of fixed tissues or cell lines, and rna extracted from ffpe is normally degraded to fewer than bp. extraction protocols a prerequisite for gene expression studies in formalin-fixed tissue samples was the establishment of a reliable and reproducible extraction method to provide detectable rnas for subsequent analysis. the quality of extracted rna can be variable among different extraction methods due to several factors, such as the contamination of rnases, proteins and genomic dna. our results showed that proteinase k digestion based protocols followed by on column dnase digestion and rna elution produced good detectable rna of ffpe. proteinase k digestion has also been demonstrated by many laboratories to be critical in rna extraction protocol possibly because of two functions. on one hand, it can degrade proteins that are covalently cross-linked with each other and nucleic acid to release rna from the matrix, thereby allowing efficient rna extraction from ffpe materials. on the other, it can inactivate rnases that tend to be stable and do not require cofactors to function. thus its activity avoids any potential reactivation of rnase during reversal of fixation in aqueous buffers. our data demonstrates that incubation in proteinase k buffer at 70°c for minutes facilitated the disruption of cross-links, resulting in improved quantity and quality of rna. the rna extracted using the modified protocols enhanced detection by a mean of cycles, which is equivalent to fold increased sensitivity. it is most likely that the additional incubation step removed the remaining cross-links between rna and protein leading to the longer rna molecules being extracted. in addition, this incubation denatured proteinase k, thus avoided its further damage to rna in the following purification procedure, which suggests the necessity of a heating application in any proteinase k based extraction of ffpe samples. taqman® preamp taqman® qrt-pcr technique is based on the 5' nuclease activity of taq dna polymerase and involves cleavage of a specific fluorogenic hybridization probe that is flanked by pcr primers. because of the small target size, many laboratories have demonstrated that it is possible to measure gene expression levels using ffpe tissues as a source of mrna. still, it seems to be problematic to perform large scale of analysis on ffpe because of the high cts and limited concentration of extracts. we employed a novel taqman® preamp technique which we found to be a practical solution to decrease ct values, and in particular suitable in our hands to generate real-time pcr results from limited amounts of input rna, such as extracted from laser captured microdissected material. the principle of taqman® preamp technique is to amplify target cdna prior to real-time taqman® pcr analysis. briefly, cdna is synthesized from total rna by use of random priming. the cdna for the specific target assays is then amplified by pre-amplification reaction using pooled gene-specific primers to increase the number of targeted copies. the pre-amplification product is diluted and finally analyzed by real-time taqman® pcr using single assay containing one pair of gene-specific primers and probe. the taqman® preamp technique addresses the challenge faced by researchers working with rare or precious samples, which only limited rna could be extracted from, to perform gene expression analyses using real-time qrt-pcr. the simple process enables the user to perform uniform amplification from as little as one nanogram of cdna or alternatively conduct up to real-time pcr reactions per pre-amplification reaction without compromising the available sample material. our results demonstrated that taqman® preamp overcame the difficulties usually caused by low yields of rna extraction from ffpe. influence of taqman® master mix and amplicon length this study demonstrated that the sensitivity of taqman® detection was influenced by choice of master mix and amplicon length in assay design. we evaluated upmm and gemm which showed similar sensitivity for short amplicons, while gemm displayed better sensitivity for longer amplicons compared with upmm in parallel snap frozen and ffpe extracts. this effect was dissipated when upmm was used after pre-amplification, possibly because of the increased copy number of template available or because of the composition of the preamp mastermix which is closer in components to gemm than upmm. reagents can have a significant effect on assay reproducibility due to some parameters such as different polymerases sensitivity, primer binding efficiency and the concentration of mg2+. karrer et al. described the monte carlo effect using plant material suggesting that the pcr reproducibility could be limited when the number of available templates is low. increased concentrations of mg2+ reduced pcr variation possibly by allowing a higher proportion of annealed primers extended by the more active polymerase. our data corroborated the observation that amplicon size is crucial in designing assays to analyze gene expression levels not only using ffpe extracts but also using rnas with high integrity. many researchers have found that short amplicons generated lower cts than longer amplicons on analysis of the same gene in ffpe. data generated in this study evaluating snap frozen samples using two sizes of gapdh demonstrated gapdh- generated a reduction in ct by – cycles over gapdh- in both gemm and preamp experiments, demonstrating that smaller amplicons give more consistent results. in addition, we found cts between ffpe and snap frozen were closer for small amplicons than that for large amplicons. for example, the analysis of gapdh using a target amplicon of bp displayed cts – cycles closer between ffpe and snap frozen than the same experiment using the longer amplicon size of bp. we further measured the amplification efficiency associated with these two sizes of gapdh assays using a broad dilution range which showed closed efficiencies with % for gapdh- and % for gapdh-. this measurement eliminates non-specific amplification that could contribute to decreased amplification efficiency of the true target. therefore, it seems reasonable to conclude that the shift of efficiency detected from frozen to fixed material is due to degradation of rna in ffpe, and logical to extrapolate that the longer an amplicon is, the more likely its template will be degraded in extracted rna. as a general rule, house-keeping genes or normalising assays should have amplicon sizes that match the size of the target whose expression is to be measured and amplicons less than bp should be employed in gene expression studies using ffpe materials.  we evaluated the effect of modifying recommended extraction protocols to reproducibly produce rna that may be successfully amplified using qrt-pcr. we have found the taqman® preamp system to be a robust and practical solution to limited quantities of rna and have demonstrated comparable results in matched ffpe and snap frozen preparations providing proof of principle that this method may reliably be utilised in the context of multiple expression analyses from individual ffpe samples. 
this study improved the quality of rna extracted from ffpe by introducing a heating step into the selected extraction protocols. modification to extraction protocols has facilitated procurement of rna that may be successfully amplified using qrt-pcr. archival formalin-fixed paraffin-embedded tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to rna degradation and modification during fixation and processing. results from the bioanalyzer and taqman® data showed improvement of rna quality extracted using the modified protocols from ffpe. further, it evaluated a novel pre-amplification system designed to enhance expression analysis from tissue samples using assays with a range of amplicon size. taqman® preamp system is a robust and practical solution to limited quantities of rna from ffpe extracts. taqman® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. taqman® preamp consistently achieved decreased ct values in both snap frozen and ffpe aliquots compared with no pre-amplification. incubation at 70°c for minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising rna integrity.
 archival formalin-fixed paraffin-embedded tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to rna degradation and modification during fixation and processing. this study improved the quality of rna extracted from ffpe by introducing a heating step into the selected extraction protocols. further, it evaluated a novel pre-amplification system designed to enhance expression analysis from tissue samples using assays with a range of amplicon size.  results from the bioanalyzer and taqman® data showed improvement of rna quality extracted using the modified protocols from ffpe. incubation at 70°c for minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising rna integrity. taqman® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. taqman® preamp consistently achieved decreased ct values in both snap frozen and ffpe aliquots compared with no pre-amplification.  modification to extraction protocols has facilitated procurement of rna that may be successfully amplified using qrt-pcr. taqman® preamp system is a robust and practical solution to limited quantities of rna from ffpe extracts. 