week 2/3 DNA technology.

What are the key differences between total RNA and mRNA?

Total RNA includes mRNA, rRNA, tRNA, and other RNAs.

But mRNA specifically has a 3’ poly-A tail.

Another difference is that mRNA carries the coding sequence for proteins; other RNAs do not.

What are the main steps in RNA extraction using column purification?

Lyse: release RNA and inactivate RNases

Bind: RNA binds silica membrane under high salt

Wash: removes contaminants

Elute: RNA released in low salt buffer for analysis

What types of RNA are found in total eukaryotic RNA samples?

mRNA (with poly-A tail), rRNA, tRNA, and other small RNAs.

How is mRNA distinguished from other RNA types during purification?

mRNA has a 3’ poly-A tail, allowing oligo-dT primers to bind and selectively copy mRNA into cDNA.

What is Northern blotting and how is it used to quantify mRNA?

Northern blotting separates RNA by size on a gel, then detects the specific RNA using a labeled probe that hybridizes to the target sequence.

How does qPCR quantify gene expression?

qPCR converts RNA to cDNA and amplifies it by PCR, measuring the amount of DNA produced during the exponential phase to quantify mRNA levels.

What are microarrays and RNA-seq used for in gene expression analysis?

Microarrays hybridize cDNA to gene-specific probes on a chip to quantify many genes at once, while RNA-seq sequences cDNA fragments to measure transcript abundance genome-wide.

What are the key experimental steps in Northern blotting?

RNA is extracted and size-fractionated on a denaturing gel. It is then transferred to a membrane. A labeled probe complementary to the target RNA is hybridized to the membrane. High-stringency washing removes non-specific binding, allowing detection of the RNA of interest.

How does qPCR quantify RNA, and what are the main chemistries used?

RNA is first converted to cDNA because PCR requires DNA templates. During PCR amplification, the amount of DNA is measured in real-time during the exponential phase. SYBR Green dye binds to double-stranded DNA, while TaqMan probes fluoresce when degraded by Taq polymerase, allowing quantification.

How do microarrays quantify gene expression?

RNA is converted into cDNA, which is hybridized to a chip containing gene-specific DNA probes. The level of hybridization reflects the abundance of the corresponding transcripts in the sample.

What are the key steps in RNA-seq and its application?

RNA is converted to cDNA, which is then sequenced to identify and quantify all transcripts present, providing a comprehensive profile of gene expression.

Why are primers needed in making cDNA and how is strand synthesis directed?

Primers are needed to initiate DNA synthesis because enzymatic extension occurs only in the 5' to 3' direction. RNA is heated with primers to allow binding and start cDNA synthesis.

What are oligo dT primers and what is their function?

Oligo dT primers are stretches of thymine bases that bind specifically to the 3' poly-A tail found only on eukaryotic mRNA, allowing selective cDNA synthesis from mRNA.

What are random primers and when are they used?

Random primers, such as hexamers or nonamers, bind at multiple sites along all RNA sequences and are used when a broad, non-specific cDNA synthesis is required.

What is a gene-specific primer and what is its limitation?

A gene-specific primer binds only to the RNA sequence complementary to it, so it produces cDNA copies of a specific target RNA but not the whole transcriptome.

the stages of PCR

Denaturation (94–95°C)

Double-stranded DNA → single strands. Breaks hydrogen bonds between base pairs

Annealing (50–65°C), Primers bind (anneal) to complementary DNA sequences

Extension (72°C) Taq polymerase synthesizes new DNA strands