I am measuring the gene expression of my two target genes for Japanese flounder namely CDO and CSD. I used 1ug of RNA to make cDNA. I took 1ul of cDNA to be used for RT-PCR and run it on gel electrophoresis. Most of the samples shows strong bands but there are one to two samples with weak bands. After confirming the presence of bands, I used my cDNA for Q-PCR. I am using SYBR GREEN for my q-PCR. I did not do dilution and used 2ul of my cDNA samples in running q-PCR.
The CT value for beta actin is around 22-32 and CT value for for CDO is 28-36 with some samples "undetermined". But for all my CSD genes, I got all "undetermined" CT values.
I would like to know if this has something to do with the concentration of my cDNA? Since I checked my primer using RT-PCR and got bands but when using the primer for q-PCR I cannot get a good result.
In addition, I tried diluting my cDNA samples 5x, 10x and 15x but still I get "undetermined" CT values. Can I add up to 5ul of my samples in running q-PCR?
When you are using primers in a q-PCR you should have carefully checked their performance. Did you? :
a) How do the amplification curves look like?
b) What is the apparent amplification efficiency?
c) What is the dynamic range for quantification?*
d) What ist the corresponding range of ct-values?
If a) is fine and b) is close to 2.0, then your result indicate that your samples do not contain enough molecules of the target sequence to be quantified.
In this case you have two options: use more cDNA (speedvac) and/or use sequence-specific primers for the RT. Another possibility is to pre-amplify the cDNA samples, but I do not recommend this for q-PCR (if you can't quantify a sequence in a q-PCR you won't get a reliable preamplification of this sequence either).
---
= range of a reasonably linear relationship between log(conc) and ct with sufficiently low variance of replicate measurements.
When you are using primers in a q-PCR you should have carefully checked their performance. Did you? :
a) How do the amplification curves look like?
b) What is the apparent amplification efficiency?
c) What is the dynamic range for quantification?*
d) What ist the corresponding range of ct-values?
If a) is fine and b) is close to 2.0, then your result indicate that your samples do not contain enough molecules of the target sequence to be quantified.
In this case you have two options: use more cDNA (speedvac) and/or use sequence-specific primers for the RT. Another possibility is to pre-amplify the cDNA samples, but I do not recommend this for q-PCR (if you can't quantify a sequence in a q-PCR you won't get a reliable preamplification of this sequence either).
---
= range of a reasonably linear relationship between log(conc) and ct with sufficiently low variance of replicate measurements.
Hi !
Depending on your machine it can display undetermined CT or NO CT.
It means that the cDNA concentration is either very weak or there is no cDNA (concentration=0).
I think your concentration is quite weak as you normally have a much lower CT with housekeeping genes cDNA...
Do you quantify your RNA concentration before doing the Reverse transcription ?
Dear Maria,
In qPCR, "undetermined" usually refers to "below detection limit" of the assay or the reactions in which the Ct value is beyond 35-36. this may be due to failed PCR reactions, may be due to presence of inhibitors, or loss of thermal uniformity in that part of the instrument block. I suggest you to run the qPCR products on an agarose gel along with some products that have given you a valid Ct value. This will indicate if qPCR reaction is working or not. In addition, you may also check by doing an amplification with the same qPCR reagents and templates on a normal PCR instrument with the same program that you are using in the qPCR instrument.
Hope this works.
Best wishes,
SD
One more thing, from your result sheet, it appears that all the samples are same (sample 1). however the Ct values vary greatly from 28 to 36. If all the reactions are replicates of the same sample, then this indicates pipetting error. You also need to fix this problem.
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