I am trying to do a conventional RT-PCR in human vaginal epithelial cells, induced with TNFα for 2h and 4h, for the gene CCL4 (MIP-1β).
We got the primer sequence from a paper Hera et al. (2009) Alteration of TLR3 pathways by glucocorticoids may be responsible for immunosusceptibility of human corneal epithelial cells to viral infections. They used it for realtime (we tried there conditions on realtime, it doesn't work for CCL3 or CCL4)
The estimated Tm of the primers are Fwd:60°C Rev:56°C, they are 20 bp each
So on conventional PCR I tried optimising the conditions to at least see if I can get it to work there.
Conditions:
1mM MgCl2 concentration
0.4μM Fwd Primer
0.4μM Rev Primer
0.4mM dNTP
using Promega GoTaq
I tried a Temp Gradient 54-61°C. 8 temp points
Below 56.5 there is a lot of non specific bands, from 54-61 I can see the product in the 2h induction, but also a band above the 1000bp mark. My product band gets lighter as the temp increases but the band seems to stay at the same intensity except at 61°C where it seams to fade a bit.
Can anyone explain this or suggest how to go about getting rid of this extra band
Hi,
If you want to compare gene expression in different treatments, you have to get rid of unwanted bands because those will interfere the amplification of your target genes.
"Below 56.5 there is a lot of non specific bands, from 54-61 I can see the product in the 2h induction, but also a band above the 1000bp mark" --> i'm confused about what you are saying here... But if the case is: from 54-61 you only saw 2 bands, one specific band for your target and one unwanted band at 1000bp, then the problem can be DNA contamination in your isolated RNA sample.
First, you have to check the primer pair if they flank a intron (I think it's likely the case). If so and you have genomic DNA in your RNA sample, then you will have a specific PCR product from cDNA of your target mRNA and another bigger PCR product from genomic DNA (cause it contains the intron).
You can check for DNA contamination in your RNA sample by running PCR with primers for a house-keeping gene on RNA sample. If yes, get rid of DNA in your RNA samples by DNase treatment.
"My product band gets lighter as the temp increases but the band seems to stay at the same intensity except at 61°C where it seams to fade a bit" --> how "lighter" your bands got? if just a little bit, then I think it may be ok, just the problem of gel running... When you use higher annealling temperature, PCR will get more specific and you may get less product. That's why your band fades a bit.
Good luck.
Duy
may be you have try with different primers, primer need to designed from two adjacent exons, excluding intron.
the cells you are using and from the published literature from where you picked up the primer are the same?
regards
aparna
The strange thing is, I did a Real-Time PCR with it using Roche SYBR Green Plus kit and it worked perfectly without any Extra band, and that is what we want to use the primers for, gene expression assay, so I do not think that it is Genomic DNA. To make sure I ran the cDNA with GAPDH on conventional PCR and there where no extra band only the GAPDH.
I tried it with the Roche SYBR Green (non-plus) Kit the first time and saw so much non-specific bands that we could not make out what is what. That is why we wanted to see if we could get it working on conventional PCR.
But thank you for all the suggestions.
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