Considerations for Extraction Stabilization and Quantification by PCR for Sensitive Sample Such as RNA

Considerations for Extraction Stabilization and Quantification by PCR for Sensitive Sample Such as RNA


  • Post By : Kumar Jeetendra

  • Source: Cole Parmer

  • Date: 10 Dec,2017

The accessibility of PCR and qPCR systems has increased to where they are almost considered a commodity product today. This is certainly the case for end-point PCR systems and some qPCR units also allowing researchers to carry out gene expression analysis more conveniently and quickly. This accessibility has been an innovative development in the community and will continue to move research forward for years to come.

There have been concerns with regards to reverse transcriptase (RT-PCR) and real-time RT-PCR analysis of sensitive sample types such as RNA due to the stability of the sample during process and storage. Just how much impact this has had upon the results may go unknown. Recent advances into techniques such as digital PCR and new advances in extraction stabilization systems have sought to address some of these concerns.


In theory, the extraction process is a relatively trivial matter: disrupt the sample’s cellular integrity and collect the released nucleic acids. However, it is never that simple. Multiple factors must be addressed.

When performing any nucleic acid extraction (especially RNA), stability and integrity of that extract must be considered. Samples being extracted in remote field studies must also take into consideration logistical concerns such as power supply, temperature control, availability to equipment, and shipping if samples are not to be tested onsite immediately.

Another concern with general tissue extracts is the heterogeneous mixture of cell states and types being sampled or extracted from. The result of performing an extraction from a mixed population of cell is apparent and will result in a gene expression profile which represents, at best, an average for that cell population.

When considering tissue extracts in diseased or infected sample against wild-type expression, care must be taken to limit the number of wild-type cells in the diseased sample as these may affect the expression profile, or the wild-type cells themselves may have an altered gene expression due to proximity to a diseased or infected cell. This has previously been documented when bulk extracts are compared with laser micro-dissected cells. Due to the above, an ability to extract smaller volumes reliably and minimize loss of target material, or in some instances perform expression profiles on one specific cell, has driven the development of single-cell gene expression analysis kits.

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