Transcript Analysis Flashcards
What is the general process of a Northern Blot
Isolate ssRNA by running a gel, transfer to a membrane, label it with probes for hybridization, then you visualize it
How is the gel transferred to a membrane?
Filter paper is placed on a tray with the transfer solution, the gel is placed on top of this. Your membrane goes directly on top of the gel with paper towels and a weight. The capillary action of the liquid will move up and the RNA will transfer to the membrane.
What are the different labels that are used in Northern Blots and how are they detected?
Radioisotopes like 32P or biotin or digoxigenin labels. Autoradiography for isotopes or antibodies/enzyme with chemiluminescence or colormetric visualization
What are some of the advantages of a Northern Blot analysis?
Can determine size of the band, blots can be stripped and reprobed, can detect mRNA in different tissues at the same time
What are some disadvantages of the Northern Blot analysis?
Not as sensitive compared to the PCR based methods, time consuming for the low output and can only use one probe at a time
What are the general steps of in situ hybridization?
You prepare tissue selections on slides by fixing them in formalin and embedding samples in parafin wax. You then hybridize by de-waxing and rehydrating, proteinase K digests and the hybridization will occur by binding to mRNA as the probe is complementary to the mRNA
What is the purpose of using proteinase K and digestion?
It increases accessibility of the molecular matrix
What is used as the control in this experiment?
The antisense strand probe is complementary to the RNA to monitor what the background information is
What is the advantage of using in situ hybridization?
You are able to visualize the localization of mRNA in the tissues
What are the disadvantages of using in situ hybridization?
hard to quantify mRNA, not very sensitive, no size information and labour intensive
What is the process of RT-PCR?
Primers are synthesized with an oligo(T) primers OR two gene specific primers, cDNA is then produced. The samples are then quantified by gel electrophoresis or fluorescence based methods
What is the disadvantage of using RT-PCR?
This is not precise and only considered a “semi-quantitative” method
What is the general process of digital PCR?
Same idea as emulsion PCR where water-in-oil droplets are produced, however some of the droplets will have no template and some will. The PCR reaction is then run and the number of droplets corresponds to the number of template molecules in the og sample. Is mostly digitized now
What are some advantages of using digital PCR?
The template copy number is absolute so you do not need a reference like in qPCR
What are the 5 different wats of making droplets?
Oil Phase Inlet, PCR mixture inlet, micro well based PCR, Channel based PCR and Printing based PCR
What is measured in qPCR?
You monitor fluorescence signal (DNA synthesis) in real time
What is the Ct value and it’s relationship to the initial template DNA?
Ct value is the number of cycles needed for the fluorescent signal to be above the threshold, there is a negative linear relationship between Ct and the logarithm of the initial template DNA concentration
How do we use qPCR to quantify the difference in gene expression levels or gene copy numbers?
You have the delta delta ct value which is the delta ct treatment gene - delta ct control gene and put a negative sign on it and put 2^ of that
What is the qPCR melting curve?
Tells you which one of the amplicants is amplified
What are the different methods of fluorescence in qPCR
dyes, probes and primers
What is a common dye used in qPCR and describe how it works
SYBR green, binds ds DNA through intercalation of the minor groove and the brightness depends on how much DNA is produced
What is fluorescence quenching and resonance transfer?
Quenching-intensity reducing
Transfer: light from one fluorophore can transfer enough energy to cause another fluorophore to emit light, but have to be close to one another
What is the Taqman signal?
Taqman is a probe with a quencher and a reporter, because Taq has exonuclease activity, the probe is displaced and the reporter is ejected and emits light. The intensity of the light relates to how much DNA is being digested
What is the molecular beacon?
Is a hairpin probe with a reporter and quencher, when annealed the reporter will fluoresce, the intensity of the light relates to how much product there is
What is the LUX primer?
A hairpin primer with a reporter that does not fluoresce as it is quenched by the hairpin. Primer becomes ss during annealing and fluoresces in the dsDNA product
What is the general process of a DNA microarray?
You make an array with each dot containing a different gene, attach a probe to the surface and label the mRNA, the probe is placed on the microarray and transcribed into cDNA
How many probes are used in a DNA microarray?
Two-one test and one control with different colours
What does the ratio of colours tell you about the samples?
If there is a lot more of one colour than the other, the gene is likely the one that is upregulated
What the general process of RNA-seq?
RNA is RT into cDNA which is then made into a library for sequencing, the number of sequence reads ofr a gene is proportional to the number of transcript in the original RNA sample
In addition to quantification, what else does RNA-seq provide abt the samples?
splicing efficiency, polymorphisms and editing sites
