Week 8 RNA Extraction and RT

as the title says

What are the steps of RNA extraction?

Lyse, homogenize, bind, wash, elute, and use in a future experiment

What 2 cultures were used as samples in the RNA extraction protocol?

E. coli + pGEM^lux- and E. coli + pGEM^lux+

What culture should have an upregulation of gadA?

E. coli + pGEM^lux+

Why was RNA (not DNA) extracted?

Since RNA represents what is actively being transcribed by the cell at the time of collection, rather than DNA, which is just a collection of genes that the cell has

Aka, by extracting RNA, we can see the genes being used at the time of extraction

How does traditional phenol extraction separate RNA?

In acidic conditions, DNA, RNA, and proteins are separated, but in more basic conditions, DNA and RNA are combined and proteins are separated

What is the most common method of separating RNA?

Acid guanidinium thiocyanate-phenol-chloroform extraction

Can the DNA polymerase used in PCR use RNA as a template?

NO

Why did we convert RNA to cDNA?

To have a template that can be used in PCR that also represents the genes that were transcribed at the time of extraction

Compare DNA and RNA polymerase?

What are the steps for reverse transcription?

1. Using the poly-A tail an oligo dT primer with a complimentary sequence is attached

2. Reverse Transcriptase is used to synthesize a single strand of DNA complementary to the original mRNA strand

3. The RNA template is then degraded by RNAse H

4. The DNA Polymerase can synthesize a double stranded piece of DNA

What are the primers used in reverse transcription?

Oligo dT primers

What enzyme synthesizes DNA complimentary to a template RNA strand?

Reverse Transcriptase

What is the result of reverse transcriptase activity?

A hybrid DNA (one strand RNA, one strand DNA)

What degrades the RNA template during reverse transcription?

RNAse H

What is reverse transcriptase PCR?

RT + PCR

Reverse transcription occurs, then PCR is conducted to amplify the synthesized cDNA

What is cDNA?

Complimentary DNA, aka (in our case) DNA synthesized from RNA

Compare and contrast RT-PCR (reverse transcriptase PCR) and conventional PCR?

PCR amplifies a region of interest using primers

RT reverse complements RNA to amplify ONLY the transcript made at a certain point

Did we conduct RT-PCR (reverse transcriptase PCR) in this experiment?

NO, we did RT and qPCR