TITLE: PCR-RFLP protocols for genotyping VEGF-A rs28357093 
AUTHORS: Caroline Christine Pincela da Costa , Nayane  Soares de Lima, Rodrigo     da Silva Santos, Angela Adamski da Silva Reis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>       The VEGF-A gene encodes a homonymous protein, responsible for regulating angiogenesis and vascular permeability. In addition, in the central nervous system, it acts as a neurotrophic factor, stimulating neurogenesis and cell survival. </span><span style = "font-style:italic;">VEGF-A</span><span> rs28357093 is located in the promoter region of the gene. There are few studies with this polymorphism, with no reports on the consequences. However, it is in a regulatory region, important for the control of gene transcription, and may influence gene and protein expression. In this document, we share our protocol for amplification and enzymatic digestion for this SNP, using the PCR-RFLP technique and silver staining for genotyping analysis.</span></div></div></div>

[STEPS]
?. [PCR amplification of the VEGF-A rs28357093 locus]
Amplication by conventional PCR using the primer sequences (Please, check note box in the end of this section for primer paper reference):Forward: 5′ - CCC CTG CCC CCT TCA ATA -3'Reverse: 5’- AGC CTC AGC CCC TCC ACA -3’ Master mix for   reactions are sufficient for PCR amplicons and RFLP genotyping analysisIt is a good recommendation include a negative control (master mix without the DNA template) for controlling contamination of the reaction.Example of one 25 µl PCR reaction:ComponentsFinal concentrationVolume10X PCR Buffer1x2.5 μLMgCl2 50mM1,0 mM1.5 μLdNTPs 10 mM Mix2.5 mM1.0 μLPrimers (Reverse and Forward Mix) 2.5 µM0.1 mM4.0 μLUltrapure Water (Milli-Q®)-13.8 μLTaq polymerase (5U/µL)1.2U/µL0.2 μLDNA template-2.0 μLTotal volume-25 μLWere used 10X PCR Buffer, MgCl2, Taq Polymerase and dNTPs from Sinapse INC ® (São Paulo - Brazil). Primers are from Integrated DNA Technologies® (IDT) (Coralville, Iowa, US).Thermal Cycler Setup StepsTemperatureTimeInitial denaturation94ºC5 minDenaturation94ºC1 minAnnealing60ºC1 minElongation72ºC1.30 minGO TO STEP 3 35X----------Final elongation72ºC7 min
Be sure to use coat, mask and gloves.Be careful when manipulate all components of the master mix, preventing contamination.
25 µl
ComponentsFinal concentrationVolume10X PCR Buffer1x2.5 μLMgCl2 50mM1,0 mM1.5 μLdNTPs 10 mM Mix2.5 mM1.0 μLPrimers (Reverse and Forward Mix) 2.5 µM0.1 mM4.0 μLUltrapure Water (Milli-Q®)-13.8 μLTaq polymerase (5U/µL)1.2U/µL0.2 μLDNA template-2.0 μLTotal volume-25 μL
StepsTemperatureTimeInitial denaturation94ºC5 minDenaturation94ºC1 minAnnealing60ºC1 minElongation72ºC1.30 minGO TO STEP 3 35X----------Final elongation72ºC7 min
VEGF-A rs28357093 primers were previously described in:Holt RCL, Ralph SA, Webb NJA, Watson CJ, Clark AGB, Mathieson PW et al. Steroid‐sensitive nephrotic syndrome and vascular endothelial growth factor gene polymorphisms. European Journal of Immunogenetics. 2003; 30(1): 1-3.
?. [Electrophoresis for check amplification]
To check amplification success, mix  of the PCR product with  of DNA loading dye. Use  of  100 bp DNA ladder (Sinapse INC® 100 bp ladder). Run on a 3% agarose (or in Polyacrylamide gel electrophoresis - PAGE) with TBE buffer. The amplified fragment must have 263 bp.
5 µl
3 µl
3 µl
If you chose for an electrophoresis in agarose and use ethidium bromide as DNA intercalantfollow the biosafety measures (wear gloves/avoid direct contact to the skin). Also, take care to not expose the eyes or skin in the UV-light when detecting the DNA bands.
?. [Enzimatic digestion and RFLP analysis for VEGF-A  rs28357093]
For RFLP, a new mix must be prepared, using the restriction enzyme HhaI (GCG^C) from Thermo Fisher Scientific®  Enzimatic digestion mix for one reactionComponentsVolumeEnzyme buffer2.0 µLHhaI restriction enzyme 10U/µL0.5 µLUltrapure Water (Milli-Q®)17.5 µLAmplicon10.0 µLTotal volume30 µLIncubate the reactions for 6 h at 37°C, with 20 min in 80ºC for enzimatic inactivation (total reaction time: 06 h and 20 min). After DNA digestion, run an electrophoresis to view the genotypes for the VEGF-A  rs28357093.Prepare a mix with   of the digests with   of DNA loading dye. Use  of 20 bp DNA ladder (BIO-RAD 20 bp ladder). Run it in a 15% PAGE with TBE buffer For a good separation, the electrophoretic run must be cover the whole length of the gel. To reveal the genotypes, use silver staining.
ComponentsVolumeEnzyme buffer2.0 µLHhaI restriction enzyme 10U/µL0.5 µLUltrapure Water (Milli-Q®)17.5 µLAmplicon10.0 µLTotal volume30 µL
10 µl
3 µl
3 µl