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From Flashcardlet > Advance Genetics Lab Test I > Flashcards

Advance Genetics Lab Test I Flashcards

1
Q

What is the ‘total’ RNA of a cell and what does it contain?

A
  • Contains all the RNA molecules found within the cell

- mRNA, tRNA, rRNA and miRNA

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2
Q

Why is RNA isolated in this practical and not genomic DNA?

A
  • The DNA will be the same in both but the RNA will be different between the control and the LBS treated samples.
  • The mRNA will be different
  • RNA is isolated as there are different levels of expression
  • DNA will have the same level of expression
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3
Q

What are RNase’s and where are they found?

A
  • Ribonuclease (RNase) enzymes rapidly degrade RNA into smaller components
  • Enzymes found in cells and tissues.
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4
Q

What precautions should be taken to prevent the effect of Rnases?

A
  • Wear gloves
  • Change gloves after touching skin, door knobs, and common surfaces
  • Using commercially available RNase-free tips, tubes, chemicals and reagents.
  • Designating bench space as an RNase free zone
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5
Q

How could the method of RNA extractions be modified to allow genomic DNA extractions?

A

Not using guanidium thiocynate (GITC).

-It denatures proteins, RNases and separates rRNA from ribosomal proteins

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6
Q

What additional RNA purification step could you carry out to purify mRNA?

A
  • Magnetic oligo beads.
  • The polyA tail would bind to the bead and then using a magnetic field, the mRNA is captured and separated from the total RNA
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7
Q

Describe the roles of GITC plays in the RNA extraction.

A

Cell are lysed by the addition of GITC which denatures proteins, RNase and separates rRNA from ribosomal proteins

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8
Q

During the phenol/chloroform step of the RNA extraction, what is found in each of the layers?

A
  • Aqueous layer: Nucleic acid (RNA)
  • Organic phase: proteins, phenol
  • Interphase: genomic DNA
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9
Q

What role does alcohol play in the RNA extraction?

A

-DNA is insoluble in alcohol, aggregating together and giving a pellet upon centrifugation

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10
Q

What is DNAse and why was it used? Why is stop solution added?

A
  • The RNA was treated with DNAse 1. It digests double and single stranded DNA into oligo and mononucleotides
  • Removes any contaminating genomic DNA which could interfere with the subsequent qPCR
  • Stop solution contains EDTA which inactivates the DNase 1 by chelating calcium and magnesium ions required for its activity.
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11
Q

Where you able to see a pellet? If so, do you think it is all RNA?

A

Yes, no its not all the RNA as not all the aqueous layer was taken

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12
Q

Describe the difference between transcriptomics and proteomics.

A

Transcriptomics

  • The transcriptome is the set of all RNA molecules, incl. mRNA, rRNA, tRNA and other non coding RNA produced in one cell or a population of cells
  • Reflects the genes that are being actively expressed at any given time

Proteomics

  • The transcriptome is the precursor of the proteome which is the entire set of proteins expressed by a genome
  • Proteomics confirms the presence of proteins and provides a direct measure of quality present
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13
Q

Describe three methods that are available for measuring gene expression

A

Northern Blotting
-RNA from a specific tissue is run out on a gel and transferred to a membrane which is then hybridised with either genomic DNA, cDNA or RNA radioactive probes to a specific gene

RT-PCR (Reverse transcription)
-mRNA is converted to cDNA using reverse transcriptase before expression in analysed.

qPCR (Real time)

  • Is an accurate and quantitative method for measuring the expression of genes relative to a housekeeping gene
  • Fluorescent dyes is added to the PCR reaction mix, binding to dsDNA, which will increase during each cycle as each time the DNA is amplified
  • Amplicon production can be measured as a function of cyclic number.
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14
Q

Describe a positive and negative aspect of using a cell line instead of a live animal.

A

Positive
-Allows experiments to be conducted that would be difficult to do within an individual

Negative

  • Cells may mutate which some may consider a new species
  • Cells maybe contaminated.
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15
Q

What do you expect to happen when we treat the HeLa cells with Lipopolysaccharide?

A
  • Increase in immune related response
  • LPS is on the outside of bacterial cells. Inflicts an immune response as it could be a bacterial infection
  • Increase in gene expression
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16
Q

What are the approximate sizes of the two RNA bands you would expect to see in the agarose gel and what do they relate to?

A

See two ribosomal bands, 60S and 40S

28S rRNA (large) about 4.5 kb
18S rRNA (small) about 2 kb
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17
Q

What is the smeary nucleic acid that runs much slower in the gel that the two RNA bands and why is it more apparent in some lanes than others?

A
  • The genomic DNA contamination

- Found in the interphase layer, easy to take up

18
Q

In the gels, what is the fast moving, very low molecular weight material that can be seen very clearly in some of the samples?

A

Degraded DNA

19
Q

What would you conclude if the ribosomal RNA bands on the gel, rather than being discrete, were difficult to see and smeared towards the low molecular weight end of the gel.

A

The RNA was partially degrading

20
Q

When using the Nanodrop, what is being measured at 230, 260 and 280 nm?

A

260 nm= RNA has a UV max at 260 nm

280 nm= proteins and phenolic compounds have a strong absorbance

230 nm= three sources of contaminants (proteins, chaotropic salts and phenol)

21
Q

What are the main reasons for converting RNA to cDNA?

A
  • cDNA is more stable and less likely to degrade quickly

- Cannot amplify RNA directly

22
Q

Why the enzyme used in the first step of RT-PCR called reverse transcriptase?

A

Its a viral enzyme, converts RNA to DNA (cDNA)

23
Q

What is the purpose of heating the reverse transcription reaction to 85 C?

A

Kills the enzyme, leaving cDNA

24
Q

Write down a reverse primer sequence which would ‘work’ with all polyA+ mRNAs?

A

TTTTT x20

25
Q

Methods also exist to reverse transcribe all RNA found within a cell (not just polyA+ mRNAs). How you could modify the reverse transcription to allow this.

A

Use a hexamer primer
-Random hexamer primer binds throughout the entire length of RNA, ensuring reverse transcription of all RNA sequences due to their random structures.

26
Q

Why might we choose to use reverse primers that are specific for the mRNA species that we intend to analyse rather than the hexamer primers?

A

Gene specific primer

  • Good when only one particular cDNA is desired (one gene)
  • It enhances sensitivity by directing all of the RT activity to a specific message rather than transcribing everything in the mix
27
Q

Describe the working principles behind the method of Real-Time PCR

A
  1. RNA is reverse transcribed using an oligo primer (can use other primers). This primer hybridises to the poly-A tail of the mRNA
  2. With the addition of the enzyme reverse transcriptase in the presence of nucleotides, produces a complementary DNA copy (cDNA) of the mRNA transcript under study
  • Measuring the expression of genes relative to a house keeping gene which are constantly being produced in all tissues.
  • Add a fluorescent dye, binds to dsDNA, increases every cycle.
28
Q

What is the difference between one step and two step RealTime PCR?

A

One step: Chuck everything into the tube that going to make cDNA and gene specific primer. Then run it.

Two step: Makes cDNA in a separate step

29
Q

What other technologies are available for measuring the accumulation of specific gene RNA transcripts?

A
  • Situ Hybridisation
  • Nuclease protection assays
  • Northern Analysis (blotting)
30
Q

Describe how the expression of the genes would change in either the control or the LPS treated cells.

  1. Interleukin-8 (IL-8)
  2. C-C chemukin receptor type 3 (CCR3)
  3. Tumour Necrosis factor alpha (TNF-a)
  4. GAPDH
A

Interleulin-8 (IL-8)

  • LPS induces IL-8 mRNA in A549 cells in a concentration dependent manner
  • Increase in expression in LBS

C-C chemokin receptor type 3 (CCR3)
-LPS up regulate proteins and mRNA expression of CCR3 in A549 cells

Tumour Necrosis Factor Alpha (TNF-a)

  • TNF-a isn’t produced in LPS treated cells but the cells do produce a wide variety of other mediators in response to LPS
  • Decrease in expression of LPS

GAPDH
-No change

31
Q

What is a normalising gene and why is it used in real time PCR? Give an example of another gene that could be used in this role.

A

Normalising genes is something that is expressed all of the time in the cell.

  • GADPH, involved in glycolysis
  • ELF1a elongation factor, eukaryotic translation
32
Q

What is the role of the Sybr green in the real time PCR reaction?

A

Fluorescent dye

-Causes fluorescent by binding to dsDNA

33
Q

What do we mean by the term “amplification of a real time PCR reaction” and what factors within the PCR can affect this?

A

DNA should double every time, so increase exponentially.

-Primer efficiency causes issues.
-Computing issues
-Pipettes have different calibrations
-Handlers errors
-How efficient PCR was
Closest to 2, the better.

34
Q

What would be the effect if the efficiency of the real time PCR was less than 2?

A

Less than 1.7, use a different primer.

  • After each cycle of PCR, product will be less than double
  • After each cycle, less DNA will be amplified.
35
Q

Before measuring the Ct values of the gene curve, what do we need to take into account?

A
  • Don’t want to include baseline fluorescence
  • Want cDNA value at exponential, not baseline crap
  • Threshold, use values above.
36
Q

What could be still done to further improve the results obtained for the Delta Delta-Ct (∆∆Ct) methods?

A
  • Test primer efficiency
  • Use standard curve, close to 2 is good
  • Use value when doing calculation
37
Q

∆Ct value

A

Ct=value obtained for a gene of interest from both a test and control sample.
∆Ct=difference between them

2^∆Ct= fold difference
Assumes that the same amount of input template is present in each sample, that the sample quality was identical and that the amplification efficiencies of the test and normal samples are the same

38
Q

∆∆Ct

2^-∆∆Ct

A

∆Ct treated- ∆Ct control

∆Ct treated= Ct treated - Ct housekeeping treated
∆Ct control= Ct control - Ct housekeeping control

2^-∆∆Ct=fold difference
Makes the assumption that the PCR efficiencies for both the normaliser and gene of interest are identical

39
Q

Primer efficiency

A

Has significant effect on calculating the relative expression of each gene, as any deviation from the theoretical efficiency of 100% (i.e. doubling every cycle) becomes compounded at each cycle, leading to a large error after 2^40 cycles.

40
Q

Pfaffl’s Method

A

Determines the change in gene expression between control and treatment compared to the house keeping gene and incorporates the actual efficiency of the primers into the calculation.

Determines the PCR efficiency by generating a standard curve.

41
Q

LPS

A
  • Are large molecules found in the outer membrane of Gram negative bacteria, which elicit strong immune responses in animal and cells.
  • Signals immune responses.

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