PCR Data Analysis & Troubleshooting 5

Question 1

In an RT-qPCR experiment, the no-RT (minus-RT) control amplifies. What is the most likely cause, and what is the fix?

Accepted Answer

Genomic DNA contamination; DNase-treat the RNA or design intron-spanning primers

Answer

Amplification without reverse transcription means a DNA template, usually genomic DNA, is present.

Question 2

A DNA sample has an A260/A280 ratio of 1.5. What does this most likely indicate?

Accepted Answer

Protein or phenol contamination

Answer

Pure DNA is about 1.8, and lower ratios suggest protein or phenol, which absorb near 280 nm.

Question 3

A nucleic acid sample has a low A260/A230 ratio (for example, 1.0). Which contaminant is most likely?

Accepted Answer

Guanidine salts or other chaotropic carryover from extraction

Answer

Guanidine salts, phenol, and carbohydrates absorb near 230 nm, which lowers the A260/A230 ratio (ideally about 2.0-2.2).

Question 4

How does digital PCR (dPCR) quantify target without a standard curve?

Accepted Answer

By counting positive partitions and applying Poisson statistics

Answer

dPCR splits the sample into many partitions and uses the fraction of positive partitions with Poisson correction to calculate absolute copies.

Question 5

What is the main purpose of the MIQE guidelines?

Accepted Answer

To define the minimum information needed to report qPCR experiments reproducibly

Answer

MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) standardizes reporting so experiments can be reproduced.

PCR - Polymerase Chain Reaction Certification Practice Test