20.02.12 RNA based techniques Flashcards
Disadvantages of using RNA for molecular testing
- 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
RNA mutation detection techniques
- Reverse transcription PCR
- Minigenes
- Northern blotting
- RNA sequencing
- Microarray
- RNAse protection assay
What can reverse transcriptase PCR be used for
- Qualitatively detect gene expression
- Quantification of RNA
- Monitoring of residual disease
- Confirm/exclude pathogenicity of variants
What happens in RT PCR
- 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.
To create cDNA what primers can be used
- 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.
Do RNA secondary structure/ lack of poly A tails affect cDNA yeild
No
Benefits to using of RT-PCR for mutation detection
- Large genes can be analysed in fewer fragments than gDNA
- Detects splicing aberrations and deep intronic variants.
Problems to using of RT-PCR for mutation detection
- Need appropriate tissue to obtain relevant RNA isoform.
- Mutations that result in premature stop codons could lead to mRNA degradation due to NMD.
CMGS BP guidelines on unclassified variants recommending using what to ensure that mRNA from both loci are represented in cDNA
SNPs or polymorphic loci.
Considerations to make when using RT-PCR to look at VUS with possible splicing effects
- 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
What gene fusion occurs in chronic myeloid leukaemia (CML)
- BCR-ABL
- Exon 13 or 14 of BCR fuses to ABL exon 2.
- Makes a 210kDa fusion protein (p210)= an abnormal tyrosine kinase
What drug can be used to block activity of BCR-ABL fusion product p210?
Imatinib
Use of RT-PCR for BCR-ABL detection
- Transcript monitoring during treatment
- prognostic indicators
Benefits of RT-PCR
- Requires small amounts of starting material
- Several methods available to suit needs
Cons of RT_PCR
- Difficult to maintain linerarity
- Sensitive to DNA contamination
- Variation in template concentration and amplification efficiency
What are minigene assays
Vector based ex vivo method used to study the effects of variants of unknown clinical significance on splicing
What can minigene assays be used for
-To elucidate cis- / trans- regulatory elements and other regulators of pre-mature RNA splicing in vivo
Steps in minigene assay
- 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.
Disadvantages of minigene assays
- May not replicate in vivo splicing patterns.
- labour intensive compared to RT_PCR
- Requires specialist facilities and equipment
What is northern blotting
- 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.
What can northern blotting be used for
- To study gene expression (compare band intensities for different tissues)
- To detect splicing isoforms, compare tissue specific variations in splice isoforms.
Advantages of N. blotting
- Able to detect small changes in gene expression
- Can detect RNA size
- Can determine alternate splicing products
- Probes only need partial homology
disadvantages of N. blotting
- 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
RNA expression arrays do what
Measure RNA to find gene expression changes
Common used for RNA expression arrays
Tumour profiling
Advantages of RNA expression arrays
-Measures mRNA transcripts of thousands of genes simulatenously.
Disadvantages of RNA expression arrays
Only known genes or ESTs (expressed sequence tags) can be analysed
WHater are miRNAs
- MicroRNAs
- Differentially expressed in tissues
What do miRNAs do
- Involved in the development of organisms
- Negative regulators of mRNA translation
- Involved in viral infection processes.
- Associated with oncogenes.
Uses of miRNA expression profiling
- Insight into gene regulation mechanisms
- Biological subgrouping, i.e. cancer biomarkers
- Identification of potential therapeutic miRNA targets
What is RNA seq
NGS sequencing of the trasncriptome
Uses of RNA seq
- Quantitative assessment of RNA
- Identification of novel transcripts including non-coding RNA, miRNA, siRNA and other small RNAs
Advantages of RNA-seq over microarray technology
- 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.
Disadvantages of RNA seq over microarray technology
- Greater bioinformatic requirements for de novo assembly to detect novel transcripts and gene fusions
- Higher cost
- Longer TAT
- Poorer quantitative accuracy
Steps in RNAseq
Isolating RNA, converting it to complementary DNA (cDNA), preparing the sequencing library, and sequencing it on an NGS platform, bioinformatic analysis.
Examples of the use of RNA based methods
- 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.
