Hello All,
I am working on E. coli and want to study its gene expression profile using qPCR. I grew the culture till stationary phase and snap freeze the cell pellet using liquid nitrogen. I isolated its total RNA using RiboEx and converted it into cDNA using iScript kit. The concentration of cDNA used for qPCR studies is 30ng/ul (undil). The reaction setup set for qPCR is the standard setup using SYBR green (10ul total volume)as mentioned in the manual. The Ct values obtained for all the genes after the run are above 35 Ct. The same results were observed even after increasing cDNA conc to 90ng/ul (Undil).
I tried all possible ways by using fresh chemicals and reagents but of no use. The RNA bands on the gel were sharp and without smear, confirming no degradation of RNA occurred. Is there any way to improve my Ct values? kindly guide me through.
Thanks in advance!
Try to use higher concentration of cDNA (200ng) you can adjust the water and cDNA volumes.
There are chances the target gene copy number is low.
Do you have 95-105% efficiency with your primers? If yes, then you just have really low expression of your genes of interest. Do you see lower Ct values for your reference genes?
Any chance you could share the amplification curves and the melt curve data of 30 ng/uL and 90 ng/uL data? Cts are mainly a function of abundance/starting amount. It seems like you put more RNA into the cDNA reaction, compare the Ct of 30 to 90 reaction. If efficiency was near 100%, you would see 1 Ct difference if you had compared 30ng vs 60ng. Here you have 3-fold difference which would correspond to roughly 1.7 cycle difference expected for 30ng vs 90ng. So if your 30ng was at Ct 35, then your 90ng should be at 35-1.7=almost 33 cycles. You should be able to see 1 cycle difference especially with technical triplicates.
How was the primer efficiency and also how is the ct for housekeeping gene?
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