How does RNA contamination affect PCR?

Genomic DNA (gDNA) contamination of RNA samples can lead to inaccurate measurement of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR).

What is PCR contamination?

What is PCR contamination? The biggest source of PCR contamination is aerosolized PCR products, which are created when you open a tube or pipette amplified PCR product. These tiny droplets easily spread all over your bench and equipment, where they can find their way into your next PCR and be amplified.

What problems can arise from PCR amplification?

Many of the common problems with PCR and RT-PCR are identified during agarose gel electrophoresis of the reaction products. These include the absence of the expected amplification product, the presence of nonspecific products, excessive smearing, and the presence of a “primer dimer” band.

How does PCR prevent cross contamination?

Separating pre- and post-amplification areas is key to preventing contamination. Prepare your PCR master mix in a template-free room (see next bullet), using reagents that never come into contact with potential sources of contamination. Maintain a separate area for analyzing PCR amplicons.

Why is contaminating DNA in our RNA purification a problem?

An important problem in measurement of mRNA levels by RT-PCR is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, or are hazardous.

How can PCR detect contamination?

4.2. The direct comparison of PCR product sequences from a sample and a control is the best way to determine whether two PCR products are similar or different. After comparison of the DNA sequence variation between the PCR products and the control, the cross contamination of samples can be detected.

How can PCR contamination be removed?

6 Ways to Minimize Contamination during PCR

1. 1) Introduction.
2. 2) Laboratory construction.
3. 3) Unidirectional Workflow.
4. 4) Pipetting Technique.
5. 5) Frequently Changing Gloves.
6. 6) Aseptic Cleaning Technique.
7. 7) Include controls in your protocol.

What is the negative control in PCR?

Both positive and negative controls are used in PCR experiments. The positive control, a known sample of parasite DNA, shows that the primers have attached to the DNA strand. The negative control, a sample without DNA, shows if contamination of the PCR experiment with foreign DNA has occurred.

How can you determine if your PCR reaction mix is contaminated with template DNA?

To check the solution for contamination, assemble negative control reactions using new reagents known not to be contaminated, and add one of the suspect solutions to each reaction. That reaction shows amplified products indicative of contamination is evidence that the solution added was contaminated.