20.02.12 RNA based techniques

Question 1

Disadvantages of using RNA for molecular testing

Answer 1

• Short half life
• Specialist extraction kits and reagents required
• Ultra-clean labs needed
• Limited expression patterns means that the diseased tissue is required, not always available

Question 2

RNA mutation detection techniques

Answer 2

• Reverse transcription PCR
• Minigenes
• Northern blotting
• RNA sequencing
• Microarray
• RNAse protection assay

Question 3

What can reverse transcriptase PCR be used for

Answer 3

• Qualitatively detect gene expression
• Quantification of RNA
• Monitoring of residual disease
• Confirm/exclude pathogenicity of variants

Question 4

What happens in RT PCR

Answer 4

• RNA species is reverse transcribed into a single stranded cDNA/ RNA hybrid.
• THe RNA is then digested and DNA polymerase generates a second DNA strand.

Question 5

To create cDNA what primers can be used

Answer 5

• Oligo-dT. Anneal to polyA tail of mRNA. cDNA population with 3’ bias. Means PCR primers that target 5’ end of transcripts may be less effective.
• Random hexamer primers. Have a random mix of the 4 bases, so bind anywhere with complimentary sequence.

Question 6

Do RNA secondary structure/ lack of poly A tails affect cDNA yeild

Answer 6

No

Question 7

Benefits to using of RT-PCR for mutation detection

Answer 7

• Large genes can be analysed in fewer fragments than gDNA

- Detects splicing aberrations and deep intronic variants.

Question 8

Problems to using of RT-PCR for mutation detection

Answer 8

• Need appropriate tissue to obtain relevant RNA isoform.

- Mutations that result in premature stop codons could lead to mRNA degradation due to NMD.

Question 9

CMGS BP guidelines on unclassified variants recommending using what to ensure that mRNA from both loci are represented in cDNA

Answer 9

SNPs or polymorphic loci.

Question 10

Considerations to make when using RT-PCR to look at VUS with possible splicing effects

Answer 10

• Need to obtain mRNA from correct tissue/ cell type
• Need to compare to a normal control (need understanding of naturally occurring splice variants and relative proportions)
• Need to rule out possibility of NMD

Question 11

What gene fusion occurs in chronic myeloid leukaemia (CML)

Answer 11

• BCR-ABL
• Exon 13 or 14 of BCR fuses to ABL exon 2.
• Makes a 210kDa fusion protein (p210)= an abnormal tyrosine kinase

Question 12

What drug can be used to block activity of BCR-ABL fusion product p210?

Answer 12

Imatinib

Question 13

Use of RT-PCR for BCR-ABL detection

Answer 13

• Transcript monitoring during treatment

- prognostic indicators

Question 14

Benefits of RT-PCR

Answer 14

• Requires small amounts of starting material

- Several methods available to suit needs

Question 15

Cons of RT_PCR

Answer 15

• Difficult to maintain linerarity
• Sensitive to DNA contamination
• Variation in template concentration and amplification efficiency

Question 16

What are minigene assays

Answer 16

Vector based ex vivo method used to study the effects of variants of unknown clinical significance on splicing

Question 17

What can minigene assays be used for

Answer 17

-To elucidate cis- / trans- regulatory elements and other regulators of pre-mature RNA splicing in vivo

Question 18

Steps in minigene assay

Answer 18

• A specific region of a gene is amplified using the patient’s DNA and a normal control i
• ligated into a plasmid expression vector, most commonly pcDNA3.1A or USR13.
• transfectedinto E.coli
• colonies (clones) containing the recombinant plasmid are identified by PCR.
• Clones are then sequenced to identify those clones which have the normal sequence and those with the variant sequence.
• DNA from each and a control empty vector are transfected into cultured human cells which are allowed to express the mingene for 24hr.
• RNA is then prepared from the cells and analysed by RT-PCR and sequencing to determine if there is a difference between the RNA (cDNA) synthesised from the normal minigene and that synthesised from the minigene with the variant sequence.

Question 19

Disadvantages of minigene assays

Answer 19

• May not replicate in vivo splicing patterns.
• labour intensive compared to RT_PCR
• Requires specialist facilities and equipment

Question 20

What is northern blotting

Answer 20

• electrophoretic separation of RNA samples by size,
• Transfer of RNA from gel to membrane
• detection with a hybridization probe complementary to part of or the entire target sequence.

Question 21

What can northern blotting be used for

Answer 21

• To study gene expression (compare band intensities for different tissues)
• To detect splicing isoforms, compare tissue specific variations in splice isoforms.

Question 22

Advantages of N. blotting

Answer 22

• Able to detect small changes in gene expression
• Can detect RNA size
• Can determine alternate splicing products
• Probes only need partial homology

Question 23

disadvantages of N. blotting

Answer 23

• Sample degradation by RNAses
• CHemicals (e.g. radioactive labels) are harmful to users
• Only looking at one or a small number of genes
• No amplification so transcripts expressed at low levels are less suitable

Question 24

RNA expression arrays do what

Answer 24

Measure RNA to find gene expression changes

Question 25

Common used for RNA expression arrays

Answer 25

Tumour profiling

Question 26

Advantages of RNA expression arrays

Answer 26

-Measures mRNA transcripts of thousands of genes simulatenously.

Question 27

Disadvantages of RNA expression arrays

Answer 27

Only known genes or ESTs (expressed sequence tags) can be analysed

Question 28

WHater are miRNAs

Answer 28

• MicroRNAs

- Differentially expressed in tissues

Question 29

What do miRNAs do

Answer 29

• Involved in the development of organisms
• Negative regulators of mRNA translation
• Involved in viral infection processes.
• Associated with oncogenes.

Question 30

Uses of miRNA expression profiling

Answer 30

• Insight into gene regulation mechanisms
• Biological subgrouping, i.e. cancer biomarkers
• Identification of potential therapeutic miRNA targets

Question 31

What is RNA seq

Answer 31

NGS sequencing of the trasncriptome

Question 32

Uses of RNA seq

Answer 32

• Quantitative assessment of RNA

- Identification of novel transcripts including non-coding RNA, miRNA, siRNA and other small RNAs

Question 33

Advantages of RNA-seq over microarray technology

Answer 33

• RNA seq does not require transcript specific probes, so an unbiased approach.
• Can detect novel transcripts
• Broader dynamic range than hybridisation technology. RNAseq quantifies discrete, digital sequencing read counts.
• RNA seq has increased specificity and sensitivity
• Easier to detect rare/ low abundance transcripts by increasing sequencing coverage.

Question 34

Disadvantages of RNA seq over microarray technology

Answer 34

• Greater bioinformatic requirements for de novo assembly to detect novel transcripts and gene fusions
• Higher cost
• Longer TAT
• Poorer quantitative accuracy

Question 35

Steps in RNAseq

Answer 35

Isolating RNA, converting it to complementary DNA (cDNA), preparing the sequencing library, and sequencing it on an NGS platform, bioinformatic analysis.

Question 36

Examples of the use of RNA based methods

Answer 36

• Assessing intra tumour heterogeneity. Causes of therapy resistance and disease recurrence. Can detect rare subclones.
• Single cell RNA seq. Good for the analysis of rare cells or heterogeneous populations.