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.
- 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.
Store undiluted DNA at –20°C in TE low buffer or ethanol.
3. Assay design and optimization
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.
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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.