methods 4

Question 1

Is Northern blot or in-situ hybridization a better approach to determine:

(A) in what cell-type(s) of the Arabidopsis root a gene of interest (GOI) is expressed

Answer 1

in-situ hybridization

Question 2

Is Northern blot or in-situ hybridization a better approach to determine:

(B) whether the rat liver or the heart has a higher level of GOI expression

Answer 2

northern blot

Question 3

Is Northern blot or in-situ hybridization a better approach to determine:

(C) the induction kinetics of a GOI in response to Drug X in the adult zebrafish brain?

Answer 3

northern blot (you could eventually follow up with in situ but northern is cheaper and faster initially)

Question 4

What is one key drawback of the reporter gene approach for studying gene expression relative to all other methods?

Answer 4

you have to make a transgenic organism

Question 5

explain RT-PCR

Answer 5

RT-PCR is a variation of PCR where the DNA template for amplification is generated by reverse transcription of mRNA

mRNA of interest is reverse-transcribed into complementary DNA (cDNA) by Reverse Transcriptase

cDNA can then be used as a template for PCR or quantitative real-time PCR (qRT-PCR)

Question 6

what is the enzyme that amplifies cDNA during rtPCR?

Answer 6

taq polymerase

Question 7

explain the different types of primers used in rtPCR (3)

Answer 7

Oligo dT – only binds to poly A tails on 3’ ends, meaning only amplifies mRNA and excludes rRNA and tRNA

random primers – primer mixture composed of random nucleobase sequences of 6, can convert all types of RNA to cDNA

gene-specific – used to amplify gene of interest if you are only interested in looking at one gene

If you’re amplifying more than one gene (even if you just need a housekeeping gene) often beneficial to go ahead and use oligo(dT) or random primers

Question 8

explain sybr green

Answer 8

sybr intercolates and binds dsDNA and fluoresces, as PCR cycles continues the fluorescent signal doubles

Question 9

explain taqman probe quenchers

Answer 9

gene specific sequence has a probe and a quencher, and intact probe and no fluorescence, when forward/reverse primers anneal the polymerase synthesizes DNA and the probe is broken down/released where it fluoresces as it leaves proximity to the quencher

Question 10

name of phenomenon quencher uses to quench reporter

Answer 10

Förster Resonance Energy Transfer (FRET)

Question 11

explain difference between primer and probe in rt-pcr

Answer 11

Probe contains the reporter/quencher, primer binds upstream of the probe and it where DNA synthesis by taq polymerase begins

During the annealing step of the PCR cycle, the probe binds to an internal region in the gene of interest, whereas the primer binds upstream of the probe

During the extension step of PCR, Taq DNA polymerase initiates DNA synthesis and extends the primer in 5’->3’ direction until it reaches the TaqMan probe

Question 12

what is a Ct value

Answer 12

The number of cycles it takes to reach a certain level of fluorescence

Question 12

How would you deal with potential contaminating genomic DNA in an RNA sample?

Answer 12

DNase (gets most DNA) + primer pairs that exlude introns (at least one should span a junction)

Question 13

how to use for rt PCR To generate an E. coli cDNA library of all genes expressed in a human or plant tissue of interest (amplified cDNAs are then subcloned into a vector and transformed into bacteria)

Answer 13

use random primer or oligo(dT) to amplify the cDNA of all expressed genes

Question 14

how to use rt-pcr To generate an Illumina RNA-seq library to study gene expression (fragmented mRNAs are adapted, reverse transcribed, and amplified)

Answer 14

use random primer or oligo(dT) to amplify the cDNA of all expressed genes

Question 15

how to use rt-pcr To generate a cDNA or cRNA probe to hybridize to a microarray, a Northern blot, or a tissue slice for an in situ to study gene expression

Answer 15

1st strand use random priming or oligo(dT) but second probe is strand specific

for a microarray you need to amplify multiple genes, even if you’re only doing a northern blot you will need at least two genes amp so might as well generate a library worth

Question 16

how to use rt-pcr To amplify and Sanger-sequence possible mRNA splicing variants for a gene of interest (i.e., to figure out how the transcript gets spliced)

Answer 16

since you don’t know where the splices are you need to do random or oligo dt

Question 17

how to use rt-pcr To amplify and subclone the cDNA for the gene of interest for expressing it in yeast (e.g., for protein purification or interaction assays)

Answer 17

can use gene specific for first step, then use oligodt or gene specific second to get full length clone of cDNA. don’t use random for second step as you’ll get fragments (random primer will start anywhere in sequence rather than 5’ end)

Question 18

how to use rt-pcr To detect the expression of a gene of interest in a tissue (to demonstrate that the gene is transcribed, ideally in a quantitative manner)

Answer 18

oligo(dt) or random because you at lease need two genes amplified bc you need a housekeeper

Question 19

how to amplify all cDNAs at once?

Answer 19

Adaptor sequences need to be added to the 5’ and 3’ ends of cDNAs

Some types of reverse transcriptase add non-template nucleotides (CCC) at the 3’ end of the cDNA 1st strand that can be leveraged for annealing a complementary GGG-containing template switching RNA oligo to enable the extension of the 3’ end of the 1st cDNA strand with a known sequence

forward primer binds the template switching oligo, reverse primer binds the oligo dt primer strandj

Question 20

explain ddPCR

Answer 20

In ddPCR, a PCR sample is emulsified in oil to produce ~20,000 nanoliter-sized droplets

Each droplet corresponds to a separate PCR amplification and is analyzed separately

The droplets are then individually counted and scored as positive or negative for fluorescence to infer the absolute number of template molecules in the starting sample using Poisson Statistics

can use taqman or sybr

Question 21

why do you rarely use strand specific primers for first round of rtPCR? (cDNA prep)

Answer 21

because there are many applications you could use the cDNA for and you may as well make it at the beginning and store it for later

Question 22

does ddPCR use DNA or cDNA?

Answer 22

it can be done with either

Question 23

explain nanostrings

Answer 23

A method to measure mRNA expression of specific genes without having to make cDNA, end up with colored bar codes

A hybridization-based method that employs two probes per gene, a reporter probe A and a capture probe B (the CodeSet)

Both probes have a region of homology (35-50 bases each) to the gene (or transcript) of interest and will specifically bind to that gene

A reporter probe carries a unique multi-color fluorescence tag that serves as a ”bar-code” or “identifier”

A capture probe is tagged with biotin that enables purification/immobilization of the gene recognized by the probe using streptavidin

Up to 800 probe pairs can be used in a single hybridization reaction, thus enabling detection and quantification of 800 genes or transcripts at once in a complex sample (e.g., a total RNA prep from a tissue of interest)