During cDNA synthesis, same amount (400 ng) of RNA was added for each sample. The volumes varied for each sample. With the synthesized cDNA we performed conventional PCR for beta- actin. When the gel was run we saw the sample in which the initial RNA volume (not conc.) was more than others had a strong band density as compared to the one in which less volume was used to synthesize cDNA. This has happened multiple times.
Can anybody tell what can be the reason?
The image is of PCR product (RNA>cDNA>PCR) plotted as:
Well 1: ladder
Well 2: PCR product with RNA volume 13.07ul
Well 3: PCR product with RNA volume 2.86ul
Well 4: PCR product with RNA volume 3.86ul
If the low volume rna is contaminated with gDNA that would lead to the low volume for 400ng but the cDNA reaction would be low yield due to the low rna amount and the low cDNA would then only produce weak pcr bands. Alternatively if you are also concentrating salts then the high salt levels might be interfering with the pcr reaction compared with the more dilute starting material
How did you determine the concentration of RNA in your samples? If you used a spectrophotometer or nano drop, you are measuring all of the nucleotides (DNA & RNA). If there is a lot of DNA in your samples, then you are getting an artificially high concentration.
Did you run out a sample of the RNA on an agarose gel to check that it's intact? Degraded RNA & intact RNA will also give the same reading with a spec.
A Qbit can differentiate DNA & RNA in a sample. Reagents are a bit expensive, but it can be worth it.
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