I am working with HUVEC cells, and tried to plate 7500, 10000,15000,and 20000 cells on 48-well plate to see how many cells are good enough for RT-qPCR.
I isolated RNA using Trizol/Qiazol method, and synthesized cDNA using NEB ProtoScript® First Strand cDNA Synthesis Kit.
I tried 10ng, 20ng, and 30ng cDNA/ PCR rxn, but they all yield high ct around 36,37 on GAPDH.
Things to check:
1) purity of your RNA: 260/230 ratio is key, here. A high 230 value represents guanidium contamination (common with trizol preps), and this chaotropic salt will interfere with cDNA synthesis. I typically reclean (just precipitate the RNA again) any samples with 260/230 ratios lower than 1.7.
2) amount of RNA used in your reaction: you never get as much from cells as you do from tissue samples, but you don't state how much RNA you're using for cDNA synthesis
3) quantity of cDNA reaction in final PCR: cDNA synthesis reaction buffer is not necessarily compatible with PCR, thus using cDNA at too high a concentration can interfere with PCR efficiency. I recommend diluting your cDNA prep at least 1/5, if not 1/10.
Given the ubiquity and high expression of GAPDH, the most parsimonious interpretation here is either that your cDNA synthesis didn't work or your PCR didn't work. Or your primers don't work: have you used these primers before successfully? Have you tried any other genes in your samples (18S, actin)?
I usually make the samples up to 500ul with DEPC/nuclease-free H2O (simply to make the volumes more manageable), add 50ul of 3M Sodium acetate (pH 5.5), 10ug of glycogen (option; increases recovery) and then add 500ul of ROOM TEMP isopropanol.
Leave at room temp for 20mins, then spin (max speed, benchtop microfuge, chilled to 4 deg if possible). Remove the supernatants, wash the pellets gently with ice-cold 75% ethanol, 2x (add 300ul 75% ethanol gently down the side of the tube, then spin again).
Finally, air dry in a fume hood or similar (to stop stuff falling into the tubes) for ~15mins, redissolve in an appropriate volume of DEPC H2O.
Assume you'll lose maybe 20% of your RNA, but the RNA you retain will be MUCH cleaner.
Hello Li
Your 260/230 ration must be more than 1.8.
As per previous suggestion of John Hildyard, isolate the RNA carefully. If problem is not resolved then you may use quigen RNA easy column isolation method for better RNA quality.
I think you may try to increase your cells upto 50000 cells.
GAPDH ct value is higher due to following reasons:
1. lower cDNA concentration (try to used 50 ng, 75 ng, 100 ng cDNA concentration )
2. lower 260/230 ratio (due to impurity reaction may not worked)
3. problem with primer or standardization of PCR condition
Wish you a best of luck for your work.
Thanking you.
Regards
Sushant Shengule
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