Pcr analysis

Pcr analysis

Good Morning,

I have a problem with qPCR measurements and I hope you can help me with that. We would like to implement a probe (TaqMan) assay.

For receiving our samples, we isolated neutrophils from buffy coat. RNA was isolated by ‘RNeasy® Midi Kit’ (Qiagen) and on-column digested with DNase I according to the manufacturer’s recommendations. Afterwards, the isolated RNA was digested with DNase I for a second time (peqGOLD DNase I, VWR) to eliminate genomic DNA. The ‘High Capacity cDNA Reverse Transcription Kit’ (Applied Biosystems) was used to convert RNA into cDNA and afterwards, ‘PrimePCR™ Probe Assay’ (Bio-Rad) or ‘Dual Labeled Probes’ and primer pairs (Eurofins Genomics) were used for quantification of transcript levels by RT-qPCR.

For the probe assay we used 12.5 ng cDNA per 10 µl per well. We performed qPCR according to that protocol: enzyme activation, 95 °C, 2 min; denaturation, 95 °C, 15 s; annealing, 60 °C, 30 s. We used the ‘GoTaq® Probe qPCR Master Mix, 2X’ (Promega) and did duplex RT-qPCR studies.

Unfortunately, our curves are very crazy (see photo). This leads to falsified values and large standard deviations. When you change the settings of the cycles to be analyzed from 3 to 40, the previously bad Cq values get better, but some Cq values that used to be good deteriorated.

We implemented several tests, including either the use of different template amounts, or different primer/probe concentrations, or different master mixes. We also performed a gradient qPCR and changed the volume per well to 20 µl. We also used non DNAse I digested samples.

But we received always more or less the same curves.

We do not have this problem using a Sybr assay with our primers that we also used in the probe assay. So maybe our probes are the problem. But what can we do to solve the problem without buying new probes?

Attached you will find our graph.

Many thanks


Источник: www.researchgate.net

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