4 replies to this topic

[hurchu]

Hi I'm now very frustrated with one step qPCR..
I have used these pair of primers for two-step before and it worked, although efficiency is only about .85.
But now I'm not even getting a single melting curve. Instead, big fluctuations, with both SYBR and ROX.
Gel shows faint band not at the right position, for one gene is larger than expected and the other gene is smaller.
And the CT of both genes are exchanged. The previous 20s moved to 30s, and the 30s to 20s.
And the no template control shows the same as 1ug reaction....

Another complicating factor is that the SYBR GreenER kit (I used for two step, 4months old, stored in 4C, which should be still working) was not working either and getting the same jungle melting curve. I'm now suspecting everything, even the machine (since the ROX fluctuates?)...

I've read some of the older posts and decide maybe do a primer titration, adding RNase inhibitor before qPCR. And I have no other ideas as by now.
Could someone give me some advice? this is way to frustrating...

Attached File(s)

•  ROX DC.bmp (1.56MB)
  Number of downloads: 27

[hurchu]

And this is the melting curve for SYBR Green..

Attached File(s)

•  SYBR DC.bmp (1.56MB)
  Number of downloads: 25

[Adrian K]

hurchu, on 30 July 2011 - 12:23 AM, said:

Hi I'm now very frustrated with one step qPCR..
I have used these pair of primers for two-step before and it worked, although efficiency is only about .85.
But now I'm not even getting a single melting curve. Instead, big fluctuations, with both SYBR and ROX.
Gel shows faint band not at the right position, for one gene is larger than expected and the other gene is smaller.
And the CT of both genes are exchanged. The previous 20s moved to 30s, and the 30s to 20s.
And the no template control shows the same as 1ug reaction....

Another complicating factor is that the SYBR GreenER kit (I used for two step, 4months old, stored in 4C, which should be still working) was not working either and getting the same jungle melting curve. I'm now suspecting everything, even the machine (since the ROX fluctuates?)...

I've read some of the older posts and decide maybe do a primer titration, adding RNase inhibitor before qPCR. And I have no other ideas as by now.
Could someone give me some advice? this is way to frustrating...

I am not very experience in qPCR, I just give my 2 cents here:
you mentioned your gel shows band not at the right position, and I suppose you use RNA as starting material: this might means you have yet to eliminate you genomic content. Also, try to change your water.

How's your positive control, negative no template control and no-RT control?

[ivanbio]

Here are some comments:

1. An efficiency of 0.85 is not good. Unless you are getting an efficiency of at least 0.98 then there is something wrong with your assay. In other words I would say your assay was never working.
2. If you are not getting the right size amplicons then you are likely amplifying something else.
3. Getting a high signal for the no template control likely means that you are measuring primers dimers. Your "SYBR_DC.bmp" file looks like primer dimers to me, or even worse multiple peaks (which is not good).

My advice is as follows:

1. Do not use one step qPCR until you have an assay that is working (with an efficiency of at least 0.98). I've never used a qPCR assay with an efficiency of less than 0.98.
2. I strongly recommend synthesizing cDNA first and using that to optimize your assay
3. I would design at least two more assays and test them all together at the same time using cDNA (two step qPCR). By comparing how well different assays, designed to detect the same gene, work compared to each other should give you an assay that works.

Good luck

[hurchu]

Thanks a lot, Adrian and ivanbio.
I did use NTC and a positive control with cDNA. Attached is what I got.
1 NTC UBQ gene
2 RNA UBQ gene
3 cDNA UBQ gene
4 NTC DDM1 gene
5 RNA DDM1 gene
6 cDNA DDM1 gene

I do realize that my previous essay are no good..
I'm now really determined to get a good essay set up. But I don't know exactly how.
Optimization includes good cycling program, primer pairs and its concentration? Is there something else I need to check?

And the pcr part, do I need to do fast two step, or normal three step? Which do you think will be better?
What do you recommend annealing temperature for the primers?
I normally use 55C, but it seems that 50C(UBQ) produce the right band in one step qPCR with RT temperature at 50C.

Attached thumbnail(s)

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Edited by hurchu, 31 July 2011 - 01:15 PM.