Skl

Hello,

I need your help with qPCR problems!
For a while now I have had problems with my real-time qPCR. The quality of my cDNA samples seems to differ so much that some samples lead to good results and some show very late or no amplifications at all.

Here are the facts:

- I am using a Spin column RNA extraction (Macherey-Nagel Kit, elution in provided RNase-free Water) to extract RNA from eukaryotic cell cultures
- gDNA is degraded on the column by DNase
- my RNA samples have 260/280 values above 2.0  ( some 260/230 values are very low but these are surprisingly not the samples with bad results)
- my RNA samples are not degraded (checked with Agilgent Bioanalyzer RNA Nano 6000 chips, RIN >9)
- all RNA samples have about the same concentrations (e.g. when extracting 9 samples all are about 180-220 ng/µL - measured in Nanophotometer)

- I am using the Revert Aid First Strand cDNA synthesis kit by Thermo Scientific (formerly Fermentas) with Oligo-dT-primers for generation of cDNA only from mRNA
- I use 1 µg of RNA for each cDNA reaction (this is whithin the recommended range of the kit)
- I take 1 µL from each cDNA sample for qPCR

- For qPCR I use the Platinum SYBR Green Mastermix by Life Technologies (Invitrogen) on the Light Cycler 480 System (Roche)
- I measure 3 housekeeping genes (Actin, Gapdh and Vezatin) and 1 target gene and prepare Mastermixes for each primer-pair
- my primers are from one exon to the next (so no amplification from gDNA possible) - the amplificate is 100 bp long
- primers are checked by regular PCR and show only one band in gel with correct size in 100 bp and melting curves in those reactions that worked are fine


Now my problems:

- Some samples show normal amplification curves for all 4 genes with Cps at about 17 for Actin
- some samples show very very late amplifictaion (e.g Cp 35 for Actin) or no amplification at all
- since the whole sample is problematic in all 4 tested genes I would think that my biological experiment is not the problem - at least the houskeeping genes should be present somehow

Since I used the same Sybr--Mastermix for all samples I conclude that the Mastermix itself and the primers are fine - the problem has to be in the steps between RNA extraction and qPCR ?!!?
I thought that maybe my cDNA concentration could be to high and therefore inhibiting the qPCR but dilutions of the cDNA templates resulted in even later amplification curves.

Please, do you have any idea or suggestion what the problem might be?
Do you know if it could be a problem with inhibitors and if so, how can I get rid of them without changing my sample concentration too much?

I am grateful for any suggestions!