Reporters are genes that are used in molecular biology to monitor expression of genes of interest
Reporters are fused to promoters, gene fragments or entire gene of interest, and their activity is assayed/analyzed
Describe GFP
often used as a reporter gene
Green fluorescent protein
beta-barrel
bright green (excited at 395nm, emission at 509nm)
Describe Luciferase
often used as a reporter gene
catalyses oxidation of the pigment luciferin and emits light
Describe Gus and LacZ
often used as a reporter gene
They encode enzymes that convert colorless substrates (x-gluc and x-gal) to blue products)
The substrate can be infiltrated into the transgenic tissues to see where the reporter gene is expressed
Reporter gene fusions: What does the following gene fusion give you information about?
A reporter fused to the promoter can provide information about when the promoter is active to transcribe the gene
Reporter gene fusions: What does the following gene fusion give you information about?
You have the native coding sequence of the protein in addition to GDP here, so you can study where the gene is transcribed, but also where the protein goes
What type of a reporter (transcriptional or translational) will you use to study:
A tissue-specific pattern of expression of a gene of interest
Transcriptional OR (Either, but ideally both)
For tissue specificity, do we have to have protein level info?- probably not.
What type of a reporter (transcriptional or translational) will you use to study:
Subcellular localization of the protein of interest
Translational
What type of a reporter (transcriptional or translational) will you use to study:
"Inducibility" by heat shock of the gene of interest
Both transcriptional and translational
What type of a reporter (transcriptional or translational) will you use to study:
Protein movement
Both transcriptional and translational.
Protein is first synthesized by ribosomes in the cytoplasm, then it can be packaged to be moved to other cells
What type of a reporter (transcriptional or translational) will you use to study:
Regulation of translation by nutrient deprivation
both: you want to see if the RNA is synthesized first, and then if the protein is created during the deprivation period.
Describe the process of Northern Blot analysis:
It is a derivative of running RNA samples on a gel
Transfer the RNA from the gel to a nylon filter via blotting
Hybridize the blot to radioactively/fluorescent labeled DNA probe for GOI
Wash and expose filter to a film
Develop the film
In situ hybridization-
a hybridization based method that enables detection and localization of endogenous mRNAs at a cellular level
What does the DIG method use for in situ hybridization?
digoxygenin is based off of a steroid from the plants of the genus Digitalis
Describe the DIG method of in situ hybridization:
DIG can be synthetically linked to dUTP nucleotides
The DIG-labeled nucleotides are incorporated into gene-specific RNA probes
Hybridized DIG-labeled probes are then detected with anti-DIG antibodies conjugated to enzyme/fluorescent dye
When would you use a [random primer] for first strand synthesis?
usually result in higher cDNA yeilds
full of randomized hexamers
bind all along the RNA transcript and overcomes many issues with secondary structure
When would you use a [oligo dt] for first strand synthesis?
binds to the 3' poly A tail of eukaryotic sample
it is a popular choice of primer for RT PCR
cannot bind to degraded or non-eukaryotic samples
When would you use a [gene specific primer] for first strand synthesis in RT PCR?
the gene specific primers will only make cDNA for the one specific target gene
good to use when you know what you're looking for
required for qRT-PCR reactions
total cDNA yeild will be lower
Why do you think short reads are not ideal for genome assemblies and a combination of short (Illumina) and long (PacBio) reads is usually used?
Short sequencing alone is not sufficient. Done in parallel with PacBio, you can get fewer gaps, and the short reads provide better coverage
With fluorescent reporters, why do you think only a few of different FPs (three or four) can be used simultaneously in the same cell?
Too many and they'll start being hard to distinguish on the spectrum
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;
-(B) whether the rat liver or the heart has a higher level of GOI expression
-(C) the induction kinetics of GOI in response to Drug X in the adult zebrafish brain?
A- in situ would be better to see with a method that gives you cellular resolution, and its hard to get enough tissue for Northern
B- Northern Blot: looking at two separate tissues and you want quantitative information. In situs are not exactly quantitative
C-Northern Blot: (simpler and cheaper to do a northern, hard to do time series in situ)
What is one key drawback of the reporter gene approach for studying gene expression relative to all other methods?
You have to be able to make a transgenetic organism to use reporter genes
For in situ hybridization, you can be somewhat quantitative within a tissue, but not between tissues
T/F
True
How is SYBR Green used in qRT-PCR?
it binds exclusively to dsDNA and becomes fluorescent upon binding.
The more dsDNA synthesized, the more dye binds and sensitive quantification can be made
What assumption can you make about the housekeeping gene?
the housekeeping gene is assumed to be expressed equally between all different conditions in different samples
(in qRT-PCR) if you see more activity of both GOI and housekeeping gene, then you just added more samples)
Describe how the fluorescent reporter probe TaqMan works
TaqMan probe is tagged with a fluorescent dye reporter (R) at the 5' end and a quencher (Q) at the 3' end
An intact probe doesn't fluoresce
In the annealing step of PCR, the probe binds to an internal region in the GOI
During extension step of PCR, Taq DNA polymerase synthesizes DNA until it hits the TaqMan probe
Then the fluorescent dye is cleaved from the probe and glows!
TaqMan reporter primer (image)
In an RT-PCR reaction, how would you deal with potential contaminating genomic DNA in an RNA sample? Why do you think DNA carryover presents a potential problem?
If you trying to purify DNA, you'll use RNA-ase to digest RNA, and vice versa if you are trying to purify RNA
There will always be some DNA in an RNA sample though
Best way to purify RNA is with PCR!
Which scenario has primers that would help to purify RNA from any genomic DNA in its sample?
Scenario 2
You want to create a signal that ignores genomic DNA
What are some possible applications of RT-PCR?
detection of expressed genes, examination of transcript variants, generation of cDNA templates for cloning and sequencing
Which primer (N6, oliogo-dt, gene-specific), would you use to apply RT-PCR in the following scenario:
"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)"
oligo-dt or N6
Which primer (N6, oliogo-dt, gene-specific), would you use to apply RT-PCR in the following scenario:
"To generate an Illumina RNA-seq library (fragmented mRNAs are adapted, reverse transcribed, and amplified)"
N6 or oligo-dt
Which primer (N6, oliogo-dt, gene-specific), would you use to apply RT-PCR in the following scenario:
"To generate a cDNA or cRNA probe to hybridize to a microarray, a Northern blot, or a tissue slice for an in situ"
oligo-dt, N6
Which primer (N6, oliogo-dt, gene-specific), would you use to apply RT-PCR in the following scenario:
"To amplify and Sanger-sequence possible mRNA splicing variants for a gene of interest"
oligo-dt
**gene-specific makes an assumption that you're going to be analyzing a particular variant of splicing, but you may have many different alternative spliced versions
Which primer (N6, oliogo-dt, gene-specific), would you use to apply RT-PCR in the following scenario:
"To amplify and subclone the cDNA for the gene of interest for expressing it in yeast (e.g., for protein purification or interaction assays)"
gene-specific and oligo-dt
** oligo-dt allows you to capture alternate splicing of hte gene
Which primer (N6, oliogo-dt, gene-specific), would you use to apply RT-PCR in the following scenario:
"To detect the expression of a gene of interest in a tissue (to demonstrate that the gene is transcribed, ideally in a quantitative manner)"
oligo-dt or N6
** Need to include N6 random primers so that you can get background information when trying to quantitate your gene's response
Describe droplet digitial PCR (ddPCR)
A PCR sample is emulsified in oil, into about 20,000 droplets
The distribution of template molecules are random
Each droplet gets PCR amplification, and if your target is in the sample, it releases TaqMan fluoresence
The droplets are counted individually as (+) or (-) for having the fluoresence
proportion containing the template molecule determined by Poisson statistics
Nanostrings
hybridization-based method that employs two gene specific probes per gene, a capture probe, and a reporter probe
both probes have homology to the gene of interest
The reporter probe carries a fluorescence tag that acts as a barcode
up to 800 probe pairs can be used in a single hybridization reaction
after the hybridization step, probe bound transcripts are purified and aligned on a cartridge
Nanostrings (image)
Microarray Procedure:
isolation of total RNA or mRNA from the tissue(s)
reverse transcription to make cDNA
labeling of the cDNA with florescent dye(s)
hybridization of the labeled probe to the DNA spots on the chip
washing the chip to remove the non-specifically bound probe
scanning of the chip to read the fluorescence intensity of each DNA spot
finally, data normalization and analysis
What technology, RT-PCR or qRT-PCR, will you use to detect alternative splicing (presence versus absence of an intron)? How will you design your primers (i.e., where would they anneal with respect to the alternatively spliced intron)?
What method, ddPCR or nanostrings, will you use to compare the expression of:
1. two genes of interest in two samples?
2. How about 100 genes in two samples?
3. How about two genes in 100 samples?
1. ddPCR
2. nanostrings
3. ddPCR
nanostrings cost the same whether you use 2 probes or 200 probes from a set
Affymetrix GeneChip Oligonucleotide Array (image)
How can you use a microarray in clinical diagnostics?
Hierarchical cluster analysis separated neuroblastoma patients into two major categories
Also, Heat maps expression of PAM genes (cancer signature genes Prediction Analysis for Microarrays)
Alternative splicing-
exons from the same gene are joined in different combinations, leading to different, but related, mRNA transcripts
Protein analysis techniques:
SDS Page
Western Blot
Immunolocalization
Affinity Purification
SDS-Page
proteins separated by size via electrophoresis.
Samples are treated to solubilize proteins and coat them with a negative charge
Western blot
immunoblotting/ a laboratory technique utilized to detect and quantify proteins in a biological sample
follows SDS-PAGE
Common secondary antibody used in Western Blotting
horseradish per-oxidase
Immunolocalization
an antibody-based detection method for proteins and other biomolecules in a cell
