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Quantitative RT-PCR
Last updated date: Mar 23, 2021 Views: 935 Forks: 0
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- cDNA is synthesized with the iScript cDNA synthesis kit (BIO-RAD).
10 µl 5 X iScript reaction mix
2.5 µl iScript reverse transcriptase
2.5 µg RNA
Add nuclease-free H2O to 50 µl total per reaction
- Load the thermal cycler to run the RT reactions.
5 minutes at 25oC
30 minutes at 42oC
5 minutes at 85oC
- Set up the SYBR Green PCR reactions using the BIO-RAD 96-well PCR plate.
5 µl 2 X iQ SYBR Green Supermix
0.5 µl forward primer (4 µM)
0.5 µl reverse primer (4 µM)
0.4 µl DNA template
3.6 µl H2O
- Seal the plate with an optical adhesive cover. Then centrifuge briefly to spin down the contents.
- Place the reaction plate in CFX96 Real-Time System (BIO-RAD).
Cycling Conditions:
1. 95oC for 15 minutes
2. 95oC for 30 seconds
3. 60oC for 1 minute (plate read)
4. GOTO 2, 39 more times
5. Melt Curve 60oC to 95oC: Increment 0.5oC for 5 seconds (plate read)
- Data are analyzed with CFX manager software (BIO-RAD). Transcript abundance is normalized against GAPDH.
- Primer sequences used in SYBR Green PCR reactions are listed in the table below:
Genes | Primer sequences |
CCN1 | Forward: 5’-TCGCATCCTATACAACCCTTTAC-3’ Reverse: 5’-GATACCAGTTCCACAGGTCTTT-3’ |
GADD45A | Forward: 5’-CCCGATAACGTGGTGTTGT-3’ Reverse: 5’-GATCAGGGTGAAGTGGATCTG-3’ |
FLG | Forward: 5’-TCAGACTCTAGTACCGCTAAGG-3’ Reverse: 5’-ACTGCCACGTGACTGTATTC-3’ |
GAPDH | Forward: 5’-GAAGGTGAAGGTCGGAGTCAAC-3’ Reverse: 5’-TGGAAGATGGTGATGGGATTTC-3’ |
HES1 | Forward: 5’-ATAGCTCGCGGCATTC-3’ Reverse: 5’-AGGTGCTTCACTGTCATTTC-3’ |
HES4 | Forward: 5’-CCTGGAGATGACCGTGAGACA-3’ Reverse: 5’-TACTTGCCCAGAACGGCG-3’ |
HES5 | Forward: 5’-TGCACCAGGACTACAGCG-3’ Reverse: 5’-GCGTGGAGCGTCAGGAACT-3’ |
ID3 | Forward: 5’-CGACATGAACCACTGCTACTC-3’ Reverse: 5’-GATGACGCGCTGTAGGATTT-3’ |
KRT1 | Forward: 5’-GGACAGCTCCTTAGCATCTTATC-3’ Reverse: 5’-GGAGTTTAAGACCTCTCCACAAA-3’ |
KRT10 | Forward: 5’-CAGTCCCAACTGGCCTTAAA-3’ Reverse: 5’-GATATCTGGGCCTGAATCTGTG-3’ |
LCE3A | Forward: 5’-GCCTGAGTCACCACAGATG-3’ Reverse: 5’-CGCCTTGCTGACCACTT-3’ |
IER5 | Forward: 5’-GGCTAACCTCATCAGCATCTT-3’ Reverse: 5’-CTCTCCTCTCCCTCCTCTTC-3’ |
IRF6 | Forward: 5’-CCCAGGGCTCGATCATTAAC-3’ Reverse: 5’-CCAGCTCATCTTCCTCATCTTC-3’ |
IVL | Forward: 5’-GCCCACAAAGGGAGAAGTATTG-3’ Reverse: 5’-GACACTGCGGGTGGTTATTT-3’ |
LOR | Forward: 5’-ACCTGGAAGAAGCCATTGAG-3’ Reverse: 5’-GTTGGGAGGTAGTTGTACAGAAA-3’ |
NRARP | Forward: 5’-GAGCTGTCCACCTGCTCCG-3’ Reverse: 5’-TCACGTTGAACTCGCAGTTGG-3’ |
RHOV | Forward: 5’-ACACCTTCTCTGTGCAAGTC-3’ Reverse: 5’-GGGAACGAAGTCGGTCAAA-3’ |
RIPK4 | Forward: 5’-CGACACCAAGCACGATGTAT-3’ Reverse: 5’-GTGCAGGATGTTCTTCTCATCT-3’ |
- Pan, L and Aster, J(2021). Quantitative RT-PCR. Bio-protocol Preprint. bio-protocol.org/prep959.
- Pan, L., Lemieux, M. E., Thomas, T., Rogers, J. M., Lipper, C. H., Lee, W., Johnson, C., Sholl, L. M., South, A. P., Marto, J. A., Adelmant, G. O., Blacklow, S. C. and Aster, J. C.(2020). IER5, a DNA damage response gene, is required for Notch-mediated induction of squamous cell differentiation. eLife. DOI: 10.7554/eLife.58081
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