qPCR Climbing to Success

Successful qPCR begins with preliminary steps of preparations.
Critical issues in the workflow need to be addressed and here, we review the entire workflow from planning and preparation phase to the real-time PCR.
We outlined the different steps in the workflow and indicated where and how critical issues can be resolved.

1. Sample processing

Important things to consider:

  • Time point of sampling, storage of samples and biological status.
  • Challenging sample types such as FFPE tissues, plants, viruses, LCM etc. require suited extraction kit.
2. Nucleic acid: Extraction, QC and Storage
  • Extraction- Select your nucleic acid extraction method using the following Selection Guide.
  • Quality control-High quality nucleic acid is important for successful qPCR. Test your RNA/DNA quality using UV Analysis or Fluorometer.
UV Analysis
RNA: OD260/280 ratio 1.9 – 2.1 (OD260 1 ~ 40 µg/ml)
DNA: OD260/280 ratio 1.6 – 1.8 (OD260 1 ~ 50 µg/ml)
 
Fluorometer
Qubit Fluorometer supplies wider range of detection (4 ng/ml – 1 mg/ml) and accurate quantitation of intact dsDNA and ssDNA in presence of RNA, proteins and free nucleotides. Assays are unaffected by the presence of phenol, salts, chloroform and ethanol.
  • Storage
Store undiluted RNA at –80 °C for 1-2 years in RNase free H2O + 0.1 mM EDTA or TE low buffer (1 mM Tris, 0.01 mM EDTA).
Store undiluted DNA at –20°C in TE low buffer or ethanol.

3. Assay design and optimization

Design your own custom primers and probe using  Custom TaqMan® Assay Design Tool or use our pre designed TaqMan™ Assays which include Pre-mixed Primers and Probe and save time and effort.

 

4. cDNA synthesis

When your starting material for qPCR is RNA you must first transcribe it into complementary DNA (cDNA) by reverse transcription from total RNA or messenger RNA (mRNA) also known as RT-qPCR. RT-qPCR can be performed in a one-step or a two-step assay.

5. Plates and covers

The volume of the PCR reaction is a significant consideration. In 96- or 384-well plates, PCR reaction volumes can be scaled down to 10 ul and 5 ul, respectively. This will not only save on reagents cost, but also allows the use of less material from your precious samples.

6. Run qPCR

Before setting up the qPCR reaction one needs to decide on how to monitor the increase in PCR products as the reaction proceeds.

Choosing the right chemistry and master mix is an important step when performing qPCR experiment. The most common detection methods when performing Real-Time PCR are SYBR and TaqMan.

In order to choose the right master mix, select the options that make up your particular experiment, in the online selection tool and the recommended Master Mix will appear.

Get advantage of 50% discount for all our SYBR mix at
sybr week 23-30.11.2020

7. Analyze qPCR results

Using  Applied Biosystems’ analysis tools.
Getting good results depends upon having a good workflow and using the right tools!
Luckily, we at Rhenium assembled all in one inclusive suite.