Sample Preparation & Nucleic Acid Extraction Flashcards

Why is DNase I treatment commonly performed on RNA extracts before RT-qPCR?

Answer: To remove contaminating genomic DNA that could produce false-positive signal

Residual genomic DNA can be amplified by primers, so DNase treatment removes it to ensure the signal reflects RNA.

What is the purpose of a no-reverse-transcriptase (no-RT) control in RT-qPCR?

Answer: To detect genomic DNA contamination in the RNA sample

Without reverse transcriptase, any amplification must come from DNA template, revealing gDNA contamination.

What is the recommended long-term storage condition for purified RNA?

Answer: -80°C, ideally aliquoted in RNase-free buffer or water

Storing RNA at -80°C in aliquots minimizes degradation from RNases and repeated freeze-thaw cycles.

Nucleic acids extracted from formalin-fixed paraffin-embedded (FFPE) tissue are best analyzed with which PCR design?

Answer: Short amplicons, typically under ~150 bp

Formalin causes crosslinking and fragmentation, so short amplicons improve amplification success from FFPE samples.

What is a major advantage of magnetic bead-based extraction over manual spin columns in a high-throughput lab?

Answer: It is easily automated without centrifugation

Magnetic beads are moved with magnets rather than centrifuges, making them ideal for automated liquid handlers.

An extraction blank (reagent-only sample processed alongside specimens) is used to detect what?

Answer: Contamination introduced during the extraction process

An extraction blank that amplifies indicates contamination from reagents, equipment, or handling during extraction.

A technician repeatedly thaws and refreezes the same RNA tube for weekly testing. What is the best corrective practice?

Answer: Divide the RNA into single-use aliquots

Single-use aliquots prevent the degradation that comes with repeated freeze-thaw cycles.