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RNA Extraction for qPCR and RT-PCR |
Reverse transcription-polymerase chain reaction (RT-PCR) is a process that transforms RNA to cDNA via a reverse transcriptase, then replicates it with a DNA polymerase. To get good results from reverse transcription, the starting RNA material must be of sufficient quantity, intact, and free of genomic DNA.
Real-time PCR—also known as quantitative PCR or qPCR—quantifies the copy number of PCR templates, such as DNA or complementary DNA (cDNA). Invitrogen kits and reagents for RNA extraction for qPCR are designed to yield the quality and quantity of RNA you need for RT-PCR reactions
Selection guide: RNA extraction kits for RT-PCR and qPCR
| Gold standard for highly pure, intact RNA | High-quality RNA in less than 20 minutes | High-throughput purification of RNA and DNA | Complete, no purification system for qRT-PCR results | |
|---|---|---|---|---|
| Type of PCR supported | Reverse transcription PCR and downstream applications | Reverse transcription PCR and downstream applications | Reverse transcription PCR and downstream applications | Straight to qPCR |
| Reagent/Kit | TRIzol reagents | PureLink kits | MagMAX kits | Cells-to-CT kits |
| Benefits | Most cited, economical | Quick and convenient | Scalable and flexible | Streamlined, best for qRT-PCR |
| Isolation method | Organic reagent | Silica filter, column or plate format | Magnetic beads (scalability, flexibility) | Chemical lysis |
| High throughput compatible | No | No | Yes | Yes |
| RT reagents included | No | No | No | Yes |
| qPCR reagents included | No | No | No | Yes |
| Prep time | ~1hr | < 20 minutes | ~45 minutes | 10 minutes |
| Compatible sample types | Most sample types, including difficult to lyse | Cells and tissues | Cells, blood, plants, tissue | Cells |
| Amount of starting material | 100 mg of tissue or 107 cells (requires 1 mL reagent) | Up to 200 mg tissue or 5 x 106 to 1 x 108 cells | Up to 100 mg tissue or up to 5 x 106 cells | 1–103 cells |
Purifying RNA samples prior to qPCR: DNA removal
RNA samples should be purified prior to qPCR. Genomic DNA contamination can lead to false positive RT-PCR results. Thermo Fisher Scientific offers a number of DNases that can be used to eliminate genomic DNA that may be present in your RNA preparation.
The DNA-free DNase Treatment and Removal Kit contains everything you need to remove contaminating DNA from RNA samples and remove the DNase without the use of Proteinase K treatment and organic extraction.
The TURBO DNase enzyme kit includes a hyperactive enzyme engineered from wild-type bovine DNase. The proficiency of TURBO DNase enzyme in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination.
Resources
Stylesheet for Classic Wide Template adjustments
For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.