qRT-PCR first timer, controls? - (May/15/2008 )

Hi everyone,

I am new to RT PCR. I am using RNAeasy to make RNA, qscript to make cDNA and I made primers using this freeware http://www.idtdna.com/Scitools/Application...nceWarning=True (specific for RT-PCR). I BLAST the product I would get and it was highly specific for just my gene of interest in my species of interest.

I didn't run the RT, a friend did using SYBR green. It worked quite nicely and I got nice reproducible (made 3 batches of cDNA from each cell line) results. HOWEVER, I didn't run any controls, and I don't know what controls to run. (normalized everything to GAPDH)

After the run I ran some of the PCR reaction on a gel and only got one product. I think this is a good sign.

The only control I've found here is to maybe run a PCR on the mRNA I made to make sure it isn't contaminated with gDNA. When I quantified the RNA after making it my ratios were quite nice (all between 1.8-2.2) so I'm assuming it is clean.

Is there anything else I can do, any big controls I'm missing?

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I am guessing a lot here about your experiment, but if you are comparing two different conditions, say erythroid cells treated and untreated with DMSO:

1. Gapdh, while used for normalization, is itself a control (loading control).
2. You take a gene known to be induced with DMSO in these cells, that is your positive control.
3. You take a gene known to be repressed by DMSO in these cells, that is your another positive control.
4. you take a gene known to be not affected by DMSO (for example, laminb1, MyoD), that is your negative control.
5. If your amplification curve slope gives right value, dissociation curve shows only one product, you are fine. no need to check OD ratio.
6. It is best to treat the RNAs first with DNAse to remove any gDNA.

hth/

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THanks for the reply. I am actually just comparing mRNA prescence between a control cell line, and the same cell line stably expressing a mutant protein. I got significant differences in expression according to the first trial, but the only thing I never checked for is gDNA.

Do you know if the RNeasy kit is prone to gDNA contaminants. If I got a good ratio would that indicate that it was fairly pure RNA?

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Yes, it is. It all depends upon your handling. Sometimes best handling still may leave you with gDNA contamination.

OD ratio 260/280 has nothing to do with gDNA contamination. It tells you about protein contamination. 260/230 tells you about solvent presence phenol, ethanol, chloroform etc. You can check gDNA contamination by running a gel, but teh best approach (for me) is to routinely digest gDNA - reduces sample to sample variation.

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Thanks for the reply.

How can you check by running a gel?

Also you digest the gDNA using DNAse after the mRNA prep? or during ( i think there is an option in the RNeasy kit)

All my replicates were very reproducible (ran triplicates, each twice)

I don't know why I'm worried, probably because it worked on the first try .

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1. Before RT-PCR: By treating sample with RNase and running on gel to see if you have high MW band (>20 Kb)
2. After RT-PCR: By running to see if you have, in addition to the product band, a gDNA band.


After RNA prep, before RT.


You are fine. As I generally tell everybody, you need to read too, in addition to getting advice. Advice may be insufficient or sometimes misleading.

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Thank you for your help and advice. I really appreciate it.

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have you designed your primers with intron-spanning sequences? because then, gDNA is not detected. but it still will screw up your OD measurements.

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I used software to do it, but I'm not sure how to tell if it is intron spanning, I know that is ideal though.

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Find you gene in the Ensembl for example (or any sequence database that displays exons) and check if the primers are within one exon or on different ones (i.e. spanning the intron).

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