Analysis of Gene Expression 2

Reverse TranscriptionWhat is the role of reverse transcriptase in?

It converts RNA into complementary DNA (cDNA), which is necessary for qPCR analysis.

Q: How does qPCR measure DNA amplification?

qPCR tracks amplification in real-time using fluorescent signals, and the Ct value indicates the cycle at which fluorescence crosses the threshold.

Q: What are the differences between SYBR Green and TaqMan probes in qPCR?

SYBR Green is cheaper but non-specific (binds any dsDNA), while TaqMan probes are more specific but costlier, using a labeled oligonucleotide.

Q: When would you choose SYBR Green vs. TaqMan probes?

Use SYBR Green for small-scale, cost-sensitive experiments and TaqMan for high-specificity, large-sample studies focused on one gene.

Q: Why are housekeeping genes used in qPCR, and how is expression compared?

They provide a constant baseline. Expression is compared using ΔCq (target - reference gene).

Q: What features are important in primer design for qPCR?

Primers should be specific, avoid dimers, span exon-exon junctions (to exclude genomic DNA), and have optimal melting temperatures.

Q: How do microarrays and RNA-Seq differ in transcriptome analysis?

Microarrays detect known sequences via hybridization, while RNA-Seq sequences all RNA transcripts, offering higher resolution and sensitivity.

Q: How is gene expression analysis used in medicine?

It identifies disease-related gene changes, aiding in cancer research, biomarker discovery, personalized medicine, and gene therapy.

What are random primers used for in reverse transcription?

Random primers are used as an alternative to oligo-dT primers to initiate reverse transcription across all RNA types, not just poly-A tailed mRNA.

How do random primers function during cDNA synthesis?

They bind at multiple sites along RNA sequences, allowing reverse transcriptase to generate short cDNA fragments from all RNA molecules.

What are common forms of random primers?

Random hexamers (NNNNNN) and random nonamers (NNNNNNNNN) are commonly used in cDNA synthesis.

What does reverse transcriptase do after binding to random primers?

It extends from the primers, incorporating complementary dNTPs to synthesize short cDNA fragments.

What is SYBR Green used for in qPCR?

SYBR Green is a fluorescent dye that binds to double-stranded DNA, allowing real-time detection of DNA amplification.

How do TaqMan probes work in qPCR?

TaqMan probes fluoresce when cleaved by the 5' nuclease activity of Taq polymerase during DNA amplification.