Gene Expression Analysis and RT-qPCR

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Questions and Answers

What is the first step in the RT-qPCR process?

Fluorescence quantification

cDNA synthesis

PCR amplification

RNA isolation (correct)

What role does reverse transcriptase play in RT-qPCR?

It quantifies the fluorescence after amplification

It converts RNA into complementary DNA (cDNA) (correct)

It amplifies cDNA during PCR

It synthesizes RNA from cDNA

Which fluorescent probe is mentioned as being used in RT-qPCR?

Texas Red

Rhodamine

SYBR Green I (correct)

Fluorescein

What is the main reason that working with RNA is more challenging than with DNA?

RNA is chemically unstable and susceptible to RNases Signup and view all the answers

During the PCR amplification step, what enzyme is primarily responsible for amplifying cDNA?

Taq DNA polymerase Signup and view all the answers

What is the purpose of the fluorescence signal generated in qPCR?

To reflect the quantity of PCR product formed Signup and view all the answers

What must be done to ensure the integrity of RNA during isolation?

Avoid contamination with RNases Signup and view all the answers

Which type of RNA is NOT typically used directly for cDNA synthesis in RT-qPCR?

Ribosomal RNA (rRNA) Signup and view all the answers

What is a critical step to minimize RNA degradation during extraction?

Extract RNA from fresh or quickly frozen samples Signup and view all the answers

Which of the following is NOT recommended for cleaning benches when working with RNA?

Bleach solution Signup and view all the answers

What is the purpose of using DEPC-treated H2O in RNA procedures?

To inactivate RNase and reduce contamination Signup and view all the answers

Which material is recommended for RNA isolation from plants?

Guanidinium isothiocyanate Signup and view all the answers

What must be done to glassware to ensure removal of RNases?

Bake at 180 to 300°C for at least 4 hours Signup and view all the answers

In the Qiagen RNeasy kit, which component is essential for inactivating RNases during the initial step?

Beta-mercaptoethanol (ß-ME) Signup and view all the answers

What does the high-salt buffer in the RNA isolation process allow?

mRNA to bind to silica membranes Signup and view all the answers

Which of the following techniques is NOT effective for reducing residual RNase activity during RNA isolation?

Autoclaving equipment Signup and view all the answers

Signup and view all the answers

Study Notes

Gene Expression Analysis

Gene expression analysis involves studying the amount of mRNA present in a cell or tissue.
Amplification curves, melt curves, and gene expression maps are used to visualize and analyze gene expression levels during various time intervals (1 hour, 12 hours, and 24 hours).
Gene expression analysis often includes techniques like RT-qPCR.

Overview of RT-qPCR

RT-qPCR is used to measure gene expression.
RNA (mRNA, tRNA, rRNA, and ncRNA, miRNA) is initially isolated.
Isolated RNA is transcribed to complementary DNA (cDNA) using reverse transcriptase (RT).
cDNA serves as a template for PCR reaction.
Fluorescent probes (such as SYBR Green I) bind to double-stranded DNA during PCR, and the generated fluorescence is quantified.
The quantity of fluorescence reflects the amount of PCR product formed.

RNA Isolation

RNA isolation is the first step in RT-qPCR.
Protocols involve cell lysis in chemical environments that denature RNases.
These chemical substances include mild detergents, phenol + SDS, or guanidinium isothiocyanate.
RNA is then fractionated from other cellular components to keep RNase activity minimal.
Commercial kits are available for RNA isolation. These kits are easy-to-use but generally more expensive compared to non-kit methods.
Using Qiagen RNeasy kits involves lysing and homogenizing samples using denaturing buffers that contain guanidine-thiocyanate and beta-mercaptoethanol (B-ME).
These procedures immediately inactivate RNases to purify intact RNA.
Ethanol is added for binding conditions.
High-salt buffers allow longer RNA molecules to bind to silica membranes.
Smaller RNAs (<200 nucleotides) are selectively excluded, making it suitable for specifically isolating mRNA.

Precautions when working with RNA

Quickly isolate RNA from samples by either using fresh ones or freezing quickly in liquid nitrogen followed by storage at -70°C.
Avoid thawing during handling.
Wear gloves to avoid contamination. Clean work surfaces with 100% ethanol.
Use specialized RNase-free plastic ware and glassware, baked at +180 to 300°C for at least 4 hours for effective inactivation of RNase.
Use DEPC-treated (Diethyl pyrocarbonate) water/solutions to prevent contamination with RNases.

Determining RNA Purity, Integrity, and Quantity

Spectrophotometer (Nanodrop) is used to determine RNA quantity and purity.
Gel electrophoresis (agarose gel) is used to ascertain the integrity of RNA.
At least 200 ng of RNA is normally loaded to visualize with ethidium bromide in a gel.
The integrity of RNA is ascertained using regular agarose gel or denaturing agarose gel specifically for Northern blot.
Fresh electrophoresis equipment must always be used.
High voltage is avoided to prevent degrading RNA during electrophoresis.

Genomic DNA contamination

RNA samples may comprise genomic DNA.
To eliminate genomic DNA contamination prior to cDNA synthesis, RNA is treated with RNase-free DNase I.
DNase I is an endonuclease that cleaves DNA nonspecifically.
DNase-free RNA is crucial in downstream applications like RT-qPCR.
Genomic DNA can interfere with RT-qPCR leading to incorrect results.

cDNA Synthesis

Reverse transcriptase (RT) converts RNA to cDNA (complementary DNA).
Oligo(dT) primers or random primers can be used.
Oligo(dT) primers specifically bind to poly(A) tails present on mRNA.
Random primers bind to any region of mRNA.

Primers

Primers are short, single-stranded DNA sequences.
Oligo(dT) primers, random primers, and gene-specific primers are used for cDNA synthesis.
Oligo(dT) primers are commonly used for eukaryotes.
Random primers are used for any RNA species.
Gene-specific primers are particularly important for amplifying specific genes of interest.

One-step vs. Two-step RT-qPCR

One-step RT-qPCR performs reverse transcription and PCR in a single tube.
Two-step RT-qPCR involves separate reactions for the reverse transcription reaction to synthesize cDNA and the PCR amplification.

Hot-start iTaq DNA pol

Antibody-mediated Hot-start iTaq DNA polymerase prevents non-specific primer binding, ensuring amplification of only the targeted gene.
This "hot-start" mechanism is critical to prevent non-specific amplification at lower temperatures.

Other Techniques for Monitoring Gene Expression

Northern blot/RNA blot.
cDNA microarray.
RNA sequencing(RNAseq).

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Description

This quiz covers the essential concepts of gene expression analysis, focusing on techniques such as RT-qPCR. You'll explore how mRNA levels are measured and the processes involved in RNA isolation and cDNA synthesis. Test your knowledge on the methods and applications of these critical molecular biology techniques.