RT-qPCR primer problem
Posted 26 May 2013 - 01:33 AM
I want to carry out absolute quantification looking at RNA copy numbers over 3 treatments. I have synthesised my RNA to cDNA. My PI designed the primers, and I tested them out on PCR and run on a 1.5% gel. Selected the best one (gel pic attached). I am now attempting to using one of my samples in standard curve (I need to quantify this using methods on previous posts). This particular sample I'm using is nice and clear on gel, but I can't seem to get any viable results on qPCR. Efficiency always over 110%, rsq value always <0.98, poor dissociation curve (variable lines). Melting curve always good.
As you can probably guess I'm a little new to qPCR. I have two questions.
Can I assume that the problem with standard curve is a primer problem?
With dissociation curve, is this just a pipetting problem? As I rarely get lines overlapping each over when doing triplicates. I also did a triplicated dilution template in parallel with a researcher who works on qPCR frequently.
I would really appreciate some help in this. If I can't get this working soon my project is dead.
Many thanks in advance
- TechnoJr likes this
Posted 26 May 2013 - 04:33 PM
This is very simple advice, but I have ruined a standard curve before because of the amount of input DNA, i.e. not flicking/mixing my 1/10 dilution enough before taking out of that tube to make my 1/100 dilution etc.
Perhaps also consider using a different sample, or maybe source a DNA standard you can purchase.
- Toby Doyle likes this
Posted 27 May 2013 - 04:28 AM
Posted 27 May 2013 - 04:36 AM
I'm using Stratagene mx3000p qPCR with SYBR green. I synthesised my RNA using Go script from Promega. I did my RNA extraction with Trizol as I have quite a few samples and limited funding unfortunately. My primer is at 179bp.
I have ran primers with DNA on PCR, no amplification on gel.
For cDNA synthesise, I used 2 uL of RNA, 1 ul random hexamare (50 uM), dNTPs (10uM). For qPCR I used SYBR green master mix and rox as reference dye with 2 uL of cDNA, making total volume of 15 uL. Also cDNA is diluted 1:5 with Depc water
I have been thawing down samples each time for primers tests on PCR and for work with qPCR. Samples are sorted at -20.
These are the problems I have come up with so far:
1. Primer melting temp - optimum round 60 degrees celsius - mine around 81
2. Poor quality template
3. Insufficient template - I have quite high Ct
4. Primer problem - redesign
Posted 29 May 2013 - 06:07 PM
But looking at your standard curve, I agree the Ct's are too high. The first three dilutions look quite well spaced, the last is letting you down. Perhaps it is too dilute (not enough input DNA) so the primers are not binding in the same cycles as the first three (i.e. primers finding it difficult to 'find' the DNA and anneal).
If you can find a different template to run your standard curve, one with a higher concentration or less degredation, I think it will look better. If possible purchase a control from one of the many heartless corporations just waiting to rip you off.
I'm sure lowering the Tm of your primers will help too.