Gene expression detection methods RNA

Dr. Ingrid Müller - AG von Laer

1 TRANSCRIPTIONAL CONTROL REGULATES DIFFERENTIATION

Four different human cells - same genes, different structures and functions due to differential gene expression

2 THE CENTRAL DOGMA

• Flow of Information: DNA Replication

Transcription Translation

A B

Cells in all living organisms are continually activating or deactivating genes through gene expression, which contain the information required for producing proteins through proteins synthesis. When a particular protein is required by the cell, the gene coding for that protein is activated. The first stage in producing a protein involves the production of an RNA copy of the gene's DNA sequence. This RNA copy is the messenger RNA. The amount of mRNA produced correlates with the amount of protein eventually synthesised and measuring the amount of a particular mRNA produced by a given cell or tissue is often easier than measuring the amount of the final protein. Levels in gene activation may vary between cancerogenic and healthy cells.

3 MOLECULAR METHODS TOOL BOX

I. Analysis II. Overexpression III. Inhibition

•Western Blot •(adding proteins to in •pharmacological inhibitors, •Proteomics vitro reactions) •dominant-negative Protein •immuno- proteins, histochemisty • protein depletion using •protein chimera antibodies •Electrophoresis •microinjection •RNAi & •Northern Blot •siRNA •RNAse protection •Morpholinos RNA assays •Microarrays •RT-PCR •RNA in-situ •Reporter genes •(microinjection) •Knock-out •Electroporation, •Integrational mutagenesis DNA (gene) •lipofection, •Classic genetics •transgenics

4 RNA METHODS: ELECTROPHORESIS

5 PURIFICATION OF MESSENGER RNA USING OLIGO DT COLUMNS

Total cellular RNA; apply at room temperature to Break open anneal polyA tail to oligo(dT) cells in the presence of RNAse Oligo(dT) inhibitors attached to AAAA.. cellulose TTTT TTTT polyA binds to 65oC Isolate RNA oligo(dT) on column

} non-polyA AAAAA.. RNA flows through polyA mRNA elutes at high temperature

6 RNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS

7 RNA METHODS: ELECTROPHORESIS

Detection using SYBR Green II: staining of ss nucleic fragments

• Denaturing of RNA to break up secondary structures • TAE / TBE running buffer • Agarose / Polyacrylamide gels

8 NORTHERN BLOTS

• Isolated mRNA separated on gel according to size • mRNA transferred to a membrane and hybridized with small number (1-5) of radioactively labeled DNA/RNA probes (35S or 32P) • Probe corresponds to gene of interest • Target RNA is spatially fixed and the labeled probe is in solution

• Reverse northern blot: Probe: isolated mRNA Substrate (fixed to membrane): DNA/RNA fragments

9 NORTHERN BLOT

10 NORTHERN BLOT

11 NORTHERN BLOT

control sample

target gene 10x

internal control gene actin, GAPDH, RPLP0 etc. 2x

Corrected fold increase = 10/2 = 5

Ratio target gene in experimental/control = fold change in target gene fold change in reference gene

How can amounts of RNA be quantified? This slide shows a virtual Northern with two lanes, one with RNA from control cells, the other with RNA from the experimental sample (eg drug treated cells). Let’s say that there is 10x the amount of signal in the experimental sample compared to the control sample for the target gene. This could mean expression of the gene has increased 10- fold, or it could mean that there is 10x as much RNA in the expt lane. To check for this one usually does a so-called ‘loading control’ in which the blot is probed for expression of a gene which does not change (e.g. actin, GAPDH, cyclophilin, RPLP0 mRNAs; ribosomaL RNA). In this case, let’s say that the loading control shows that there is twice as much RNA in the expt lane. Thus the real change in the target gene is 10/2 =5 fold. We can express this in a more general fashion: ratio targ et g ene ( experim ental/control) = fold chang e in targ et g ene ( expt/control)

fold chang e in reference g ene ( expt/control)

12 NORTHERN BLOT

Measure relative expression levels of mRNA

1. mRNA isolation and purification 2. electrophorese on a gel 3. The gel is probed by hybridizing with a labeled clone for the gene under study.

northern blotting of human tissues

13 RNA METHODS: NORTHERN PRO’S AND CON’S

Pros – Established and widely accepted method for single mRNA species detection – Newer techniques can be done without radiation (fluorescence)

Cons – Same semi-quantitative limitations as seen in SDS- PAGE/Western Blots for protein – Time consuming – Low throughput

Perform 35,000 Northerns to monitor expression of all genes!!!

14 MICROARRAYS

15 FINGERPRINTS OF GENE EXPRESSION ⇒ MICROARRAYS

Normal Cell

Cancer Cell

16 MICROARRAY

Collection of microscopic DNA spots attached to a solid surface (glass, plastic, silicon chip) forming an array for the purpose of expression profiling, monitoring expression levels for thousands of genes simultaneously

Applications: Analysis of expression patterns in: – Tissues – Disease states

Sub-typing complex genetic disease e.g. cancer

17 DNA ARRAY TECHNOLOGY

Spot Density Array Type Probe Target Labeling (per cm 2 ) Nylon Macroarrays < 100 cDNA RNA Radioactive Nylon Microarrays < 5000 cDNA mRNA Radioactive/Flourescent Glass Microarrays < 10,000 cDNA mRNA Flourescent Oligonucleotide Chips <250,000 oligo's mRNA Flourescent

Fabrication Printing using fine-pointed pins on glass slide Photolithography

18 PHYSICAL SPOTTING

19 PHOTOLITHOGRAPHIC SPOTTING

20 TWO POPULAR MICROARRAYING PLATFORMS

Spotted microarrays Commercially available Oligonucleotide microarrays

Probes: Affymetrix “Gene Chips” Synthesized oligos (70 mer) Probes: cDNA Oligos (25 mer),represent gene fragments Small PCR products (500-1,000bp) Produced by photolithography on silica Corresponding to mRNAs matrix >10,000 probes 500,000 probes

21 SPOTTED MICROARRAYS

tumor normal

Pro

Mixing Only one chip needed per experiment

Con Absolute gene expression levels cannot be measured

22 GENECHIPS® BY AFFYMETRIX

cDNA

23 GENECHIPS® BY AFFYMETRIX

Single nucleotide polymorphism (SNP): responsible for genetic variation and the source of susceptibility to genetically caused diseases

SNP microarrays: particular type of DNA microarrays used to identify genetic variation in individuals and across populations

Applications: Forensics Measurement of genetic predisposition to disease Profiling somatic mutations in cancer

24 MICROARRAY “Heat map”

Here we can see an annotated close-up of an affymetrix chip, with the regions relating to several genes highlighted.

25 GENECHIPS® BY AFFYMETRIX

26 THE PROBLEM

NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION

THE SOLUTION

• PCR: - most sensitive - can discriminate closely related mRNAs - technically simple - but difficult to get truly quantitative results using conventional PCR

Real time PCR was developed because of the need to quantitate differences in mRNA expression. PCR methods are particularly valuable when amounts of RNA are low since the fact that they involve an amplification step means they are more sensitive.

27 RT-PCR REVERSE TRANSCRIPTASE-PCR

• RNA containing virus – PCR doesn’t work on RNA templates Extract RNA from cells

– RT PCR RNA • make cDNA copy of RNA sequence first

• PCR the cDNA copy of RNA PCR

Can observe very low levels of expression Requires very small amounts of mRNA

Have to design multiple custom primers for each gene

28 RT-PCR

Measures relative expression of mRNA

1. Isolate and purify mRNA 2. reverse transcription 3. PCR amplification 4. run on gel

First, the mRNA’s are isolated and purified. Next, the mRNA is reverse transcribed, possibly using a gene-specific primer. Recall that transcription normally makes an RNA copy from a DNA template. This is reverse transcription, as it is making a DNA copy from an RNA template. Next, standard PCR is used to amplify the number of copies of the transcript under study. Finally, the resulting product is run out on a gel and probed.

29 PCR

Conventional 100 PCR t n u o m A

t c u d o r P

0 Cycle number 35

Regular PCR involves performing the reaction and electrophoresing the final product – not reflective of starting amount

30 WHAT’S WRONG WITH AGAROSE GELS?

* Poor precision * Low sensitivity * Short dynamic range < 2 logs * Low resolution * Non-automated * Size-based discrimination only * Results are not expressed as numbers * Ethidium bromide staining is not very quantitative

31 REAL TIME PCR

Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

• kinetic approach • early stages • while still linear

Real time PCR is a kinetic approach, where you look at the reaction in the early stages while it is still linear. There are many real time machines available. This is the one we use (the BioRad Icycler IQ real time PCR instrument). The lid slides back and then we put samples in a 96-well plate format inside, so one can look at a lot of samples simultaneously. The machine contains a sensitive camera which monitors the fluorescence in each well of the 96-well plate at frequent intervals during the PCR reaction. In our case, as DNA is synthesized, more SYBR green will bind and the fluorescence will increase.

32 REAL-TIME qPCR

• What is real-time quantitative PCR?

A PCR-based method to measure the number of copies of a particular DNA fragment in a given sample

- Amplification products are labeled by a DNA binding dye or probe chemistry that emit fluorescent signal when excited. -The signal strength of the emitted light is directly proportional to the amount of PCR product in the reaction -The fluorescence intensity is detected and recorded every cycle

DNA amplification is monitored as the reaction occurs

33 REAL-TIME PCR

• Real-Time PCR based on PCR (1983, Kary Mullis)

• PCR thermocycler, UV lamp, camera and EtBr

• EtBr integrated in ds DNA is detected by UV

Advantages Real-Time PCR

• Reproducible DNA- und RNA quantification • No electrophoresis • Large dynamic area (up to 9 Logs) • High throughput

34 QUANTITATIVE PCR (qPCR)

Conventional 100 PCR t n u o m A t c u

d Realtime o r qPCR P

0 Cycle number 35

35 REAL-TIME QPCR VS.TRADITIONAL PCR

Starting e c

Template n e c

PCR s e Round 1 r o u l 2X F Template Cycle Real-time analysis End-point analysis Detection and constant Agarose gel for product PCR monitoring of amplification detection Round 2 products is possible during the entire run. Analysis and quantification can be made in the logarithmic 4X phase of the reaction rather Template than at the end of the reaction. 30-40 more cycles

36 PRINZIP DER TAQMAN-PCR

+ specific - expensive

37 SYBR GREEN

• Second method of detection of PCR product Fluorescence low – Detecting an increase in fluorescence intensity

• Utilize a fluorophore that emits light only when complexed with dsDNA

• Increasing amount of dsDNA product with each round of amplification gives a Fluorescence high corresponding increase in fluorescence

+ unexpensive - unspecific

In this presentation, we will be using Sybr green to monitor DNA synthesis. Sybr green is a dye which binds to double stranded DNA but not to single- stranded DNA and is frequently used to monitor the synthesis of DNA during real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does, which is why we use Sybr Green rather than ethidium bromide; we also use Sybr green because the ratio of fluorescence in the presence of double-stranded DNA to the fluorescence in the presence of single-stranded DNA is much higher that the ratio for ethidium bromide). Other methods can also be used to detect the product during real-time PCR, but will not be discussed here. However, many of the principles discussed below apply to any real-time PCR reaction.

38 iCYCLER (BIORAD)

39 QUANTITATIVE PCR (QPCR)

Create a standard curve with 10-fold serial dilutions of PCR product – assign arbitrary values

Compare values from standards with values for unknown sample

100 Plateau information is not quantitative

STD 1: 1,000,000 STD 2: 100,000 STD 3: 10,000 STD 4: 1,000 STD 5: 100 STD 6: 10

Product Amount Sample: 6,592 0 Cycle number 35

40 STANDARD DILUTION

Ct value

41 REAL-TIME PCR ADVANTAGES

* not influenced by non-specific amplification * amplification can be monitored real-time * no post-PCR processing of products (high throughput, low contamination risk) * ultra-rapid cycling (30 minutes to 2 hours) * wider dynamic range of up to 1010-fold * requirement of 1000-fold less RNA than conventional assays (3 picogram = one genome equivalent) * detection is capable down to a 2-fold change * confirmation of specific amplification by melting point analysis * most specific, sensitive and reproducible * not much more expensive than conventional PCR (except equipment cost)

42 PCR BIAS: LIMITATIONS TO QUANTIFICATION

• Some genes may amplify more readily • Some cycles may be more efficient • A reagent may become limiting in the reaction

43 REAL-TIME PCR DISADVANTAGES

* setting up requires high technical skill and support * high equipment cost

* * * * intra- and inter-assay variation * Littte RNA stability * DNA contamination (in mRNA analysis)

44 RNA-QUALITY CONTROL: AGILENT BIOANALYZER (LAB-ON-A-CHIP)

To control the quality of RNA, researcher can use the Bioanalyzer Agilent Technologies which is a microfluidic Lab-on-a-Chip technology. You can have a qualitative and quantitative analysis of RNA in 1 hour (one Agilent Chips included 12 RNA Samples).

Capacity optimale : 1 chip/ hour (12 RNA sample)

45 INTACT TOTAL RNA

50 45 Distinct 18S Distinct 28S ribosomal 40 ribosomal subunit subunit (or prok. 23S): (or prok. 16S) 28S 35 ideally 2X size of 18S 30

25 18S 20

Fluorescence 15 Marker 10 ~100 bp 5 S S 8 8

0 1 2

19 24 29 34 39 44 49 54 59 64 69 Time (seconds) Marker

46 HEAVILY DIGESTED RNA

47 COMPLETELY DIGESTED RNA

~100 bp 175

150

125

100 e c n e c s

e 75 r o u l Marker F 50

25 S 8

0 1

19 24 29 34 39 44 49 54 59 64 69 Time (seconds)

48 A PROPER LADDER

200 bp

30

25

25 bp 20 2 kb 500 bp 1 kb 4 kp 15 ~6 kb

10 Fluorescence

5

0

19 24 29 34 39 44 49 54 59 64 69 Time (seconds)

49 RNA METHODS: RNASE PROTECTION

50 RNA METHODS: RNASE PROTECTION

51 RNA METHODS: RNASE PROTECTION

Pros – Medium through-put (~ 10 genes) – Good reproducibility allow statistical analysis of tests – Cost-effective

Cons – Gel-based – 2-3 days to complete

52 COMPARISON OF QUANTITATIVE ASSAYS

Sensitivity Dynamic Range 100 Real-Time PCR 108 101 107 102 106 103 105 104 104 105 103 106 Microarrays 102 107 RPA 101 108 Northern 100

53 RNA IN SITU HYBRIDISATION

54