TITLE: The estimation of the relative gene expression in the hypothalamus and liver of agouti mice using quantitative reverse transcription PCR
AUTHORS: Kira V. Derkach, Irina O. Zakharova, Inna I. Zorina, Andrey Bakhtyukov, Irina V. Romanova, Liubov V. Bayunova, Alexander O. Shpakov

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Female C57Bl/6J mice have a mutation in the A</span><span style = "vertical-align:super;">y</span><span> locus associated with overexpression of ASIP1, an antagonist of the types 3 and 4 melanocortin receptors. This mutation is characterized by the development of the melanocortin-type obesity. After treatment of the agouti mice with metformin (MF) in the different doses for 10 days, the hypothalamus and liver sections of wild-type mice, untreated agouti-mice and MF-treated agouti-mice were dissected, and the expression of the genes encoding the components of the hypothalamic signaling systems and the liver proteins were estimated by a quantitative reverse transcription PCR (qRT-PCR). The total RNA was isolated using ExtractRNA Reagent. The reverse transcription reaction was carried out using MMLV RT kit. The polymerase chain reaction was performed using the qPCRmix-HS SYBR + LowROX, and the amplified signals were detected with the Applied Biosystems® 7500 Real-Time PCR System (“Life Technologies, Thermo Fisher Scientific Inc.”, Waltham, Massachusetts, USA). The primers for the genes of signal proteins were selected using the Primer-BLAST web application. The genes encoding 18S rRNA and hypoxanthine-guanine phosphoribosyl transferase (Hprt) were used as an endogenous control. Relative expression was calculated using the ddCt method compared with the control group. </span></div><style>
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[STEPS]
?. [Preparation of samples]
Total RNA was isolated from the sections of the hypothalamus and liver using the ExtractRNA Reagent (TRIzol analogue) (“Evrogen”, Moscow, Russia) according to the manufacturer‘s protocol. (http://evrogen.com/)
?. [Preparation of samples]
The samples containing 1 μg of RNA were reverse-transcribed to cDNA using the MMLV RT kit (“Evrogen”, Moscow, Russia) and the random oligodeoxynucleotide primers were used according to the manufacturer‘s protocol. (http://evrogen.com/)
?. [Preparation of reaction mixture in the 96-well plate]
The 51µl of distillated water add to a 200 µl PCR-tube.
?. [Preparation of reaction mixture in the 96-well plate]
Add 15µl of qPCRmix-HS SYBR+LowROX ("Evrogen", Moscow, Russia).
?. [Preparation of reaction mixture in the 96-well plate]
Add 6µl of the forward and reverse primer mixture until 0.4 µМ of each.
?. [Preparation of reaction mixture in the 96-well plate]
Add 3µl sample containing 30 ng of cDNA or distilled water in control sample.
?. [Preparation of reaction mixture in the 96-well plate]
Mix the solution by pipetting and quick spin.
?. [Preparation of reaction mixture in the 96-well plate]
Dispend the PCR tube containing the reaction mixture (75 µl) into three wells (25 µl in each) in the 96-well plate. Cover the plate with the seal for qPCR plates.
?. [Preparation of reaction mixture in the 96-well plate]
Briefly mix and centrifuge 96-well plate in the centrifuge/vortex for the PCR plates and confirm that reaction mixture is at the bottom of the wells, with no bubbles.
?. [Preparation of reaction mixture in the 96-well plate]
Place the 96-well plate into the Applied Biosystems® 7500 Real-Time PCR System (“Life Technologies, Thermo Fisher Scientific Inc.”, Waltham, Massachusetts, USA).
?. [PCR analysis]
qRT-PCR amplification continues about 2 hours.
?. [PCR analysis]
The melting curve analysis should show the presence of a single peak and the absence of the primer dimers in each reaction containing the sample.
?. [Data analysis]
Data analysis is performed using the 7500 Software v2.0.6 and the Expression Suite Software v1.0.3. All the data are presented as relative mRNA level of the target gene (RQ). The calculation of the expression level of the target gene produced by the method of ΔΔCT (Schmittgen, Livak, 2008): ΔCT =CT gene of interest – CT endogenous controlΔΔCT = ΔCT sample or control – ΔCT control meanRQ= 2-ΔΔCTTo calculate the RQ values for the control and experimental groups, the mean ΔCT control is calculated, which is subtracted from all the control and experimental ΔCT.
?. [Preparation of samples]
In the end of experiment animals were decapitated, and the hypothalamus and liver sections were removedand frozen immediately at -80°C before the analysis and then used for qRT-PCR
?. [PCR analysis]
The following qPCR amplification protocol is used: an initial denaturation at 95 °C for 5 min; a three-step amplification andquantification program consisting of 38 cycles of 95 °C for 30 s, 55–58 °C for10 s (annealing temperature depends on the specific pair of the primers), and 72 °C for 30 s; and the ABI Melt Curve program to verify the presence of a single peak and the absence of primer-dimer formation in eachtemplate-containing reaction. The expression of each gene from all the studied groups of animals is determined on the same plate.  ABCDEF1  <span lang="EN-US" style="font-size:10.0pt;
  mso-ansi-language:EN-US;mso-fareast-language:RU">No    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Gene    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Localization    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Primer    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">NCBI Reference Sequence  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Temperature of annulling 2  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">1    Pomc<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;;
  mso-ansi-language:EN-US;mso-fareast-language:RU">CAGTGCCAGGACCTCACC<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    NM_008895.4    55  3  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">CAGCGAGAGGTCGAGTTTG    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   4  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">2    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Agrp    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    ACAACTGCAGACCGAGCAGAA<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    NM_001271806.1<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">58  5  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    CGACGCGGAGAACGAGACT<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   6  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">3    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Npy    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    CGGGAGAACAAGTTTCATTTCC<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    NM_023456.3<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    56  7  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    ACAGAAAACGCCCCCAGAAC<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   8  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">4    Lepr<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">GCATGCAGAATCAGTGATATTTGG    NM_146146.2    56  9  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">CAAGCTGTATCGACACTGATTTCTTC    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   10  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">5    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Mc3r    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">CAAGGAGATTCTCTGCGGCT    NM_008561.3<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    56  11  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">TCCCGTCTGAGCGTTGTTTT    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   12  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">6    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Mc4r    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">GGGTCGGAAACCATCGTCAT    NM_016977.4<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    56  13  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">TGCAAATGGATGCGAGCAAG    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   14  7    Bax  For 5’–3’  TGGAGCTGCAGAGGATGATTG    NM_007527.3    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">56  15          Rev 5’–3’  GAAGTTGCCGTCAGAAAACATG         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   16  8    Bcl2  For 5’–3’  GTGGATGACTGAGTACCTGAAC    NM_009741.5    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">55  17          Rev 5’–3’  GAGACAGCCAGGAGAAATCAA         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   18  9    IL1beta  For 5’–3’  TCCAGGATGAGGACATGAGCAC    NM_008361.4    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">56  19          Rev 5’–3’  GAACGTCACACACCAGCAGGTTA         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   20  10    TNFalpha  For 5’–3’  TATGGCCCAGACCCTCACA    NM_013693.3    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">56  21          Rev 5’–3’  GGAGTAGACAAGGTACAACCCATC         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   22  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">11    Hprt<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">AGCCGACCGGTTCTGTCAT    NM_013556.2    57  23  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">GGTCATAACCTGGTTCATCATCAC    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   24  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">12    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">18S rRNA    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;background:white;mso-ansi-language:EN-US;
  mso-fareast-language:RU">GGGAGCCTGAGAAACGGC    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">NR_003278.3<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    57  25  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;background:white;mso-ansi-language:EN-US;
  mso-fareast-language:RU">GGGTCGGGAGTGGGTAATTT    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">
ABCDEF1  <span lang="EN-US" style="font-size:10.0pt;
  mso-ansi-language:EN-US;mso-fareast-language:RU">No    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Gene    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Localization    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Primer    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">NCBI Reference Sequence  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Temperature of annulling 2  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">1    Pomc<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;;
  mso-ansi-language:EN-US;mso-fareast-language:RU">CAGTGCCAGGACCTCACC<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    NM_008895.4    55  3  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">CAGCGAGAGGTCGAGTTTG    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   4  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">2    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Agrp    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    ACAACTGCAGACCGAGCAGAA<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    NM_001271806.1<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">58  5  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    CGACGCGGAGAACGAGACT<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   6  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">3    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Npy    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    CGGGAGAACAAGTTTCATTTCC<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    NM_023456.3<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    56  7  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    ACAGAAAACGCCCCCAGAAC<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   8  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">4    Lepr<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">GCATGCAGAATCAGTGATATTTGG    NM_146146.2    56  9  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">CAAGCTGTATCGACACTGATTTCTTC    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   10  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">5    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Mc3r    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">CAAGGAGATTCTCTGCGGCT    NM_008561.3<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    56  11  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">TCCCGTCTGAGCGTTGTTTT    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   12  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">6    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Mc4r    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">GGGTCGGAAACCATCGTCAT    NM_016977.4<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    56  13  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">TGCAAATGGATGCGAGCAAG    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   14  7    Bax  For 5’–3’  TGGAGCTGCAGAGGATGATTG    NM_007527.3    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">56  15          Rev 5’–3’  GAAGTTGCCGTCAGAAAACATG         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   16  8    Bcl2  For 5’–3’  GTGGATGACTGAGTACCTGAAC    NM_009741.5    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">55  17          Rev 5’–3’  GAGACAGCCAGGAGAAATCAA         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   18  9    IL1beta  For 5’–3’  TCCAGGATGAGGACATGAGCAC    NM_008361.4    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">56  19          Rev 5’–3’  GAACGTCACACACCAGCAGGTTA         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   20  10    TNFalpha  For 5’–3’  TATGGCCCAGACCCTCACA    NM_013693.3    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">56  21          Rev 5’–3’  GGAGTAGACAAGGTACAACCCATC         <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   22  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">11    Hprt<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">AGCCGACCGGTTCTGTCAT    NM_013556.2    57  23  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">GGTCATAACCTGGTTCATCATCAC    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">   24  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">12    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">18S rRNA    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">For 5’–3’    <span lang="EN-US" style="font-size:10.0pt;background:white;mso-ansi-language:EN-US;
  mso-fareast-language:RU">GGGAGCCTGAGAAACGGC    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">NR_003278.3<span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">    57  25  <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">Rev 5’–3’    <span lang="EN-US" style="font-size:10.0pt;background:white;mso-ansi-language:EN-US;
  mso-fareast-language:RU">GGGTCGGGAGTGGGTAATTT    <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">     <span lang="EN-US" style="font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language:
  RU">