RNA-Based Methods

Question 1

General applications of RNA based analysis in diagnostics

Answer 1

• Detect and characterise breakpoints of gene fusions (e.g. BCR-ABL)
• Detect aberrant splicing – allowing for detection of deep intronic sequence changes that DNA sequencing of might miss – if using Exon sequencing
• Can sequence coding regions with fewer fragments if primers not positioned within introns

Question 2

General disadvantages of using RNA vs DNA

Answer 2

• Short half life
• Specialist extraction kits and reagents required
• Ultra-clean laboratory areas required
• Limited expression patterns may mean that the required tissue is not available for analysis

Question 3

What are the key techniques for utilising RNA for mutation detection?

Answer 3

1. Reverse transcription polymerase chain reaction (RT-PCR)
2. Minigenes
3. Northern blotting
4. RNase protection assay
5. RNA sequencing

Question 4

What is the principle of Reverse transcription polymerase chain reaction (RT-PCR)?

Answer 4

• All RNA species in a cell (mRNA, rNA etc) can be reverse transcribed into single stranded cDNA / RNA hybrid that is more stable than RNA, by first strand cDNA synthesis
• The RNA strand can then be digested and DNA polymerase used to generate a second DNA strand
• For cDNA creation Oligo-dT primers and/or random hexamer primers can be used.
• Oligo dT primers anneal to the polyA tails of mRNA- can lead to a cDNA population with a 3’bias, compromising the effectiveness of PCR primers that target the 5’end of transcripts.
• RNA transcripts lacking a 3’polyA tail will not be reverse transcribed.
• Random Hexamer Primers (random mixes of the 4 bases) bind anywhere the complimentary sequence is found.
• cDNA yield not affected by problems with RNA secondary structure/lack of polyA tails.

Question 5

What Key considerations when performing RT-PCR mutation detection?

Answer 5

• Difficult to get RNA from the appropriate tissue where the gene of interest is expressed and in the correct isoform.
• For example cDNA sequencing of the DMD gene can be used to detect intronic variants affecting splicing within its large introns or inversions.
• To sequence the muscle specific isoform Dp427 need mRNA from a muscle biopsy which is invasive and often not possible.
• Some tissues may have very low levels of mRNA = hair
• Mutations that result in premature stop codons could result in degradation of the resulting mRNA due to nonsense mediated decay. CMGS best practice guidelines on unclassified variants recommend using known SNPs or polymorphic loci to e_nsure mRNA from both loci are represented_ in the cDNA.

Question 6

What are Mini-Gene assays?

Answer 6

• Vector based ex vivo method that can be used to study the effects of variants of unknown clinical significance on splicing.
• Minigenes are used to elucidate cis-regulatory elements, trans-regulatory elements and other regulators of pre-mature RNA splicing in vivo.
• Minigenes have been applied to the study of a diverse array of genetic diseases due to the abundance of alternatively spliced genes and the specificity and variation observed in splicing regulation.

Question 7

Describe the plasmid vector construct used in a minigene assay

Answer 7

• Exon(s)/intron(s) of interest inserted into a splicing reporter (USR13), which has non gene specific exons from COL2A.
• The fragment containing the region of interest is ligated into an expression vector (pcDNA3.1/myc/His).
• The construct contains a CMV promoter that will trigger the expression of the region of interest.

Question 8

Describe the protocol of a minigene assay

Answer 8

1. Transfect normal, mutant and vector control DNA into HEK293T cells.
2. Harvest cells and extract RNA.
3. Perform RT-PCR to compare mutant and normal transcripts for differences.
4. Sequence to confirm.

Question 9

what are the disadvantages of RT-PCR?

Answer 9

• Whilst it doesn’t require patient samples - mutations can be inserted by site directed mutagenesis, however, they may not replicate in vivo splicing patterns.
• More laborious than RT-PCR from patient samples and most diagnostic labs do not have access to the necessary facilities to perform the assay.

Question 10

What are ‘expression arrays’?

Answer 10

RNA Microarrays. Measuring RNA in order to find gene expression changes, commonly used for expression profiling in tumours.

Question 11

Advantages and disadvantages of ‘expression arrays’?

Answer 11

• Advantages
  
  • Measuring mRNA transcripts in order to determine the gene expression profile in the sample
  • Monitoring thousands of genes simultaneously
• Disadvantages
  
  • Only known genes or ESTs (expressed sequence tags) can be analysed

Question 12

What is ‘RNA-seq’?

Answer 12

• Also called whole transcriptome shotgun sequencing (WTSS)
• Uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment in time

Question 13

What are the advantages of RNA-seq vs RNA arrays?

Answer 13

1. Unbiased detection of novel transcripts:
2. Broader dynamic range
3. Increased specificity and sensitivity
4. Easier detection of rare and low-abundance transcripts

Question 14

What are the advantages of RNA arrays over RNA-seq?

Answer 14

1. Lower costs
2. Short turn-around time
3. Quantitative accuracy
4. Ease of data generation
5. Easier bioinformatic analysis/requirements