1. BioSci 203 lecture 23 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 23 - mRNA analysis Bruce Blumberg (blumberg@uci.edu) –office - 4203 McGaugh Hall –824-8573 –lab 5427 (x46873), 5305 (x43116), 5345 BSII –Office hours W 1-2 or by appointment –Office hours will be held on 3/5 and 3/12 –The next six lectures will be held in Tamkin building room F114 Today –mRNA Analysis Techniques to detect and quantitate mRNA –Northern –RNase protection –RT-PCR –in-situ hybridization Transcript mapping

2. BioSci 203 lecture 23 page 2 © copyright Bruce Blumberg 2001. All rights reserved Analysis of genes and cDNAs Characterization of cloned DNA –what things do we want to know about a new gene? Complete DNA sequence –cDNA sequence –genomic sequence? –Restriction enzyme maps? Where are introns and exons? –Particularly if knockouts are coming where and when is mRNA expressed? –How abundantly is it expressed in each place? –Is there any association between expression levels and putative function? where is the promoter(s)? –Alternative promoter use? –Mapping transcription start(s) What is the function of this gene? –Loss-of-function analysis decisive »knockout »antisense »mutant mRNA e.g. dominant negative –gain of function may be helpful »transgenic »mutant mRNA - constitutively active transcription factor

3. BioSci 203 lecture 23 page 3 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA What are some important things we wish to know about mRNAs? –How large? –How many transcripts? –Where is it expressed? –When is it expressed? –Expression levels and abundance? –Is it responsive to various stimuli serum hormone treatment drug treatment PO 4 modulators –is it relatively stable or unstable?

4. BioSci 203 lecture 23 page 4 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - size and splicing mRNA size determination - only one way - Northern –availability of good RNA size markers allows reasonably good sizing –which to use, poly A + or total RNA? A + much more sensitive (50-100x) –what about mRNAs with no or short tails? total much simpler –gel limitations - 20 μg is practical limit –what is a key factor in sizing mRNAs?

5. BioSci 203 lecture 23 page 5 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - size and splicing (contd) mRNA size determination (contd) –how to determine whether size differences result from alternative polyadenylation sites alternative splicing alternative promoter usage? –Few simple ways available isolate and characterize a lot of cDNAs –carefully compare sequences and restriction maps –where are the differences? »3’ end suggests polyadenylation »internal suggests splicing »5’ suggests promoter use »possibilities are not mutually exclusive

6. BioSci 203 lecture 23 page 6 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - size and splicing (contd) mRNA size determination (contd) –what is origin of size differences (contd) mapping of cDNA vs mRNAs –requires a systematic scan of the entire cDNA about 500 bp at a time –nuclease mapping is simplest »prepare ss probe from subclone or PCR with phage polymerase promoter attached »hybridize to mRNA »digest with nucleases, e.g., RNase, S1, mung bean, etc. »run denaturing gel and compare fragment pattern with restriction map of cDNA mapping of cDNAs to genomic organization –requires good genomic map or sequence –do cDNAs hybridize to different parts of genomic sequence? »Suggests alternative splicing »or promoter usage

7. BioSci 203 lecture 23 page 7 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation Quantitation of mRNA levels –possible methods Northern analysis nuclease protection RT-PCR –What do all of these methods measure rate of transcription? Rate of degradation? Northern analysis –advantages both size and abundance can be measured blots can be reprobed with many genes –disadvantages stringent for integrity of RNA may be difficult to detect multiple bands in a single blot requires a significant amount of RNA –may require A+ RNA for sensitivity –semi-quantitative if normalize to abundance of other mRNAs only gives you relative levels requires that reference RNA levels do not vary much between tissues

8. BioSci 203 lecture 23 page 8 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Quantitative Northern analysis (contd) –can be absolutely quantitative if an internal standard is used e.g. compare with a standard curve of known synthetic mRNAs –not typically done this way –nuclease protection assays are more frequently used advantages – is simultaneous detection of size and abundance –reprobing disadvantages –may need a lot of RNA –difficult to detect multiple bands/blot

9. BioSci 203 lecture 23 page 9 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Quantitation of mRNA levels –Northern analysis (contd) what are some good internal standards to use for quantitative comparisons? –housekeeping genes »β-actin »GAPDH »cyclophilin –none of these seem to be truly similar in all tissues shown

10. BioSci 203 lecture 23 page 10 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Quantitation of mRNA levels –Northern analysis (contd) what are some good internal standards to use for quantitative comparisons? –18S rRNA? Are rRNAs reasonable controls? –rRNAs represent 90-99.5% of total RNA –if two samples differ 5 fold in A + RNA how will this be accounted for? »Sample 1 - 0.5% A +, 99.5% rRNA »sample 2 - 2.5% A +, 97.5% rRNA –can these samples be reasonably normalized to rRNA? What to use? –Probably best to normalize to A + RNA

11. BioSci 203 lecture 23 page 11 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Nuclease protection assays –approach hybridize a SS cDNA to RNA sample –probe must be larger than protected region digest remaining single stranded regions electrophorese on denaturing PAGE –advantages less sensitive to slightly degraded mRNA absolutely quantitative can tolerate large amounts of RNA (100+ μg) –allows detection of rare transcripts –but gives high background multiple simultaneous detection –disadvantages more tedious than Northern no blot to reuse multiple simultaneous detection is very difficult to optimize

12. BioSci 203 lecture 23 page 12 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Nuclease protection assays (contd) –early techniques used SS-DNA probes and S1 or mung bean nuclease in vivo labeled M13 DNA primer extended cDNAs –RNase protection is now the dominant flavor of the assay widespread availability of bacteriophage RNA polymerases (T3, T7, SP6) and cloning vectors –easy to make high specific activity probes –or huge quantities of RNA in vitro commonly used enzymes –RNase I - cuts after all 4 bases »cloned »strong preference for ss-RNA »enzyme of choice –RNase A - cuts 3’ of U and C residues »not cloned, must be highly purified from bovine pancreas –RNase T1 - cuts 3’ of G »not cloned »often combined with RNase A

13. BioSci 203 lecture 23 page 13 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Nuclease protection assays (contd) –absolute quantitation make a standard concentration curve with synthetic mRNA compare with RNA from tissue can estimate from std curve or use Phosphorimager or similar instrument to get precise quantitation

14. BioSci 203 lecture 23 page 14 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Nuclease protection assays (contd) –relative quantitation claimed that up to 12 probes can be detected simultaneously –very difficult to optimize –one key factor is specific activity of probes »high SA for rare targets »low SA for abundant or reference RNAs once optimized this is the best way to quantitate multiple mRNAs requires careful probe design and construction one probe must be “invariant” reference RNA

15. BioSci 203 lecture 23 page 15 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR - reverse transcriptase mediated PCR –approach reverse transcribe mRNA -> cDNA amplify with specific primers quantitate –flavors relative quantitation –as with NPA - compare with reference RNAs absolute quantitation –by comparison to synthetic reference –competitive PCR –various fluorescent dye mediated methods –advantages very fast very simple works with tiny amounts of material –limitations efficiency of RT reaction is not identical for all mRNAs easy to fall outside of linear amplification range –many investigators never even bother to determine where linear range is! Errors increase exponentially with amplification

16. BioSci 203 lecture 23 page 16 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR reverse transcriptase mediated PCR –relative concentration determination perform multiplex reaction using two primer sets –1 for reference –1 for experimental advantages –very simple –no fancy equipment required disadvantages –careful attention to linear region for both primer sets –often must add one set during reaction »companies claim to have products that eliminate the need for this –more than 2 primer sets are not reliable

17. BioSci 203 lecture 23 page 17 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR –absolute concentration - competitive PCR approach –both reference and target use same primer set –same efficiency –different sized fragments

18. BioSci 203 lecture 23 page 18 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR (contd) –absolute concentration - competitive PCR (contd) Approach (contd) –titrate in synthetic RNA and determine where band intensity is equal »this is the concentration of target »What is wrong in the example below?

19. BioSci 203 lecture 23 page 19 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR (contd) –absolute concentration determination - competitive PCR (contd) advantages –precise quantitation –fast –very sensitive disadvantages –requires careful optimization and validation to ensure identical efficiency –need to make reference RNA with internal deletion to get different sized fragment

20. BioSci 203 lecture 23 page 20 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR (contd) –absolute concentration determination real time PCR Taqman, molecular beacons –Fluorescent methods that allow direct quantitation of PCR product approach –make a special oligonucleotide that has a fluor and a quenching group on it. »When whole, no fluorescence –perform PCR reaction, if primer anneals, Taq polymerase removes the reporter group which can now fluoresce

21. BioSci 203 lecture 23 page 21 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR (contd) –absolute concentration determination - Taqman, etc Fluorescence detected continuously in real time advantages –can be detected in real time with proper instrument –no difficulties with linearity –multiplexing of probes possible (limited by available dyes) –very good for clinical diagnostics disadvantages –requires instrument »varies from expensive to extremely expensive »Not of equal quality –need to make custom oligos - can be expensive –must know something about relative abundance of mRNAs before setting up reactions –careful optimization required for best results »primer concentrations »target concentrations

22. BioSci 203 lecture 23 page 22 © copyright Bruce Blumberg 2001. All rights reserved RT-PCR (contd) –absolute concentration determination – Sybr Green Alternative real time RT- PCR utilizes a single dye approach –Extend a single template –Detect ds DNA with a specific dye Analysis of mRNA - quantitation (contd)

23. BioSci 203 lecture 23 page 23 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) RT-PCR (contd) –absolute concentration determination – Sybr green Plot lift off time Generate standard curve

24. BioSci 203 lecture 23 page 24 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) –RT-PCR Sybr Green (contd) Advantages –No special primers needed –Single dye, simple –Fast, robust and quantitative –Good for routine use Disadvantages –Need instrument –Single dye, can’t multiplex –Problems with multiple fragments »Melting curves required –Absolute quantitation requires std curve

25. BioSci 203 lecture 23 page 25 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - quantitation (contd) Which RT-PCR method to use? –Small number of primer sets used many times? Taqman type –Need to multiplex primer sets? Taqman type –Large number of primer sets few times each? Sybr Green

26. BioSci 203 lecture 23 page 26 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - in situ hybridization In situ hybridization –a variation of the nuclease protection assays –approach prepare non-radioactive RNA probe with tagged nucleotide –digoxygenin –biotin –dig >>> biotin hybridize to target –tissue sections –whole embryos –chromosomes digest unhybridized probe with RNase detect the hybridized probes with an antibody directed against the tag –anti-digoxygenin –streptavidin perform histochemical reaction to detect the enzymatic assay associated with the antibody –typical enzyme is alkaline phosphatase –typical substrate is 5-bromo-4-chloro-3- indoyl phosphate (BCIP) - deep blue color –many color variations now available -green, salmon, magenta

27. BioSci 203 lecture 23 page 27 © copyright Bruce Blumberg 2001. All rights reserved Analysis of mRNA - in situ hybridization (contd) In situ hybridization (contd) –advantages sensitive in situ - therefore can localize mRNAs in intact organism only method to detect mRNAs at single cell resolution possible to sequentially detect different probes –disadvantages NOT QUANTITATIVE time consuming

28. BioSci 203 lecture 23 page 28 © copyright Bruce Blumberg 2001. All rights reserved Promoter mapping Have gene, have cDNA, where is the promoter(s) –transcription start sites need to be mapped to delineate promoter boundaries –two approaches nuclease mapping primer extension really need to use both together –nuclease mapping cannot distinguish between introns and untranslated regions nuclease mapping of genomic clone protected by mRNA can locate potential exons in genomic sequence –primer extension can precisely determine transcription start site synthesize a primer to the most 5’ part of a cDNA or exon prepare labeled 1st strand cDNA using this primer - primer runoff if the fragment size is <500 bp or so, use the primer to sequence a genomic clone in parallel with a primer runoff assay –can map to the nucleotide where start site is if the fragment size is > 500 bp then the precise start can’t readily be determined using this primer –get another one

29. BioSci 203 lecture 23 page 29 © copyright Bruce Blumberg 2001. All rights reserved Promoter mapping (contd) –Alternative splicing vs different promoter use once start site(s) is mapped for a transcript one can ask whether transcripts differing at 5’end use this start site or another one –if a different start site one must conclude a different promoter. It is relatively common to use the same promoter but splice differently at 5’ end to get different mRNAs but it is also common to use two different promoters

30. BioSci 203 lecture 23 page 30 © copyright Bruce Blumberg 2001. All rights reserved Identification of mutations Identification of mutations in populations –various gel-based methods exist that compare sequences in non-denaturing gels SSCP - single stranded conformational polymorphism analysis limited to analysis of mRNA/cDNAs –brute force sequencing more tedious than cDNA sequencing but cDNAs are not often available from living patients may be only reasonable choice for large numbers works best when hotspots can be targeted exons and introns can be sequenced –sequence polymorphism analysis RFLP (restriction fragment length) SNP (single nucleotide polymorphism) –http://www.ncbi.nlm.nih.gov/SNP/ –heritable polymorphisms occur ~ 1500 bp –these can be mapped and used to generate a crude map of the human genome »1.6 million validated, non-redundant SNP clusters exist at the moment –these can be used for many purposes »tracking genes in families and populations »locating hotspots