Methodologies 2002: RNase Protection Assay

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Methodologies 2002RNase Protection Assay

• Lisa Hazelwood
• Pharmacology
• Sanders-Bush Lab

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Outline of Discussion

• What, when and whyutility of the RPA

• Assay overview

• Pros and cons

• Optimizing Troubleshooting

• Similar techniques

• Examples from the literature

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What is an RNase Protection Assay(or RPA)?

• In Brief It is a method used to determine the
• levels of mRNA expressed in tissues of
• different origin.

• Utility of this technique spans many
  disciplines
• it may be used as a primary or secondary
  method
• of detection.

• It is considered the gold standard for both
• detecting and quantifying RNA.

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When is the RPA Commonly Used?
Examining the different expression profiles of

• Different types of tumors or tumors in different
• tissues.

• Drug treatment versus control tissues (i.e.,
• antidepressants).

• Developing tissues at different stages.

• And many, many more!!!

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Why is the RPA so useful?

• It is extremely sensitive
• The probe can be synthesized with high levels
• of radioactivity to increase signal
• Low-level transcripts can be easily detected

• It is very specific
• The probes can be specifically designed to
• minimize cross-reactivity with other transcripts
• Stringency can be readily adjusted with
• temperature changes

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An Overview of the Assay

• Probe synthesis
• Sample RNA isolation
• Denaturing and hybridization of probe and target
• Digestion of single-stranded RNA
• PAGE analysis

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Probe Synthesis
In vitro transcription of high specific
activity, antisense probe
T3
NOTE there are no unlabeled Uridines!!
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Probe Purification
Gel purification of probe
Need autorad or secondary method for
detection

• If possible, quantify the specific activity of
  the probe
• so you can add an excess

• A radiolabeled probe can NOT be stored long-term

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Obtaining Total RNA
Be sure your conditions Are RNase free!!!
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Obtaining the mRNA
Heat denature total RNA
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Bringing the pieces togetherDenaturing
Hybridization
Combine so that probe is the limiting reagent
Sample
Denaturation and hybridization both take place at
elevated temperatures and have specific buffer
requirements
Probe
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RNase Digestionof Unprotected Fragments
The double-stranded probe-target hybrid is
protected from RNase A digestion
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PAGE Analysis of Protected Fragments
Precipitate
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Controls

• Use a sense RNA probe
• This will not hybridize to the target and will
  prove that youre
• detecting what you think you are!!

• Keep one tube RNase free
• This will ensure that you did not contaminate
• your sample (you will see a large, undigested
  probe)

• Probe with an internal standard
• This shows even loading of samples controls for
  technique

• Use control mRNA
• You can select mRNA that will hybridize with your
  control probe
• but not your test probe

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A Real World ExampleHow does drug treatment
influence gene expression
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Synthesize and Purify Probes
T3
Target probe Glutamate receptor
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The Analysis
Denature, hybridize and digest the samples
Antidepressant treatment increases glutamate-R exp
ression
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Important Points Helpful Hints

• Probe must be in excess
• You can change the quantity or specific activity
  of the probe
• and detect changes in the protected fragment
  intensity
• You can change the quantity of sample mRNA and
  detect changes
• in the protected fragment intensity

• You can assay for multiple transcripts in one
  sample
• Be sure that the protected probes will be
  different lengthsthis
• goes for your internal standard probe,
  too!!

• You can adjust the stringency to fit your needs
• Decrease the stringency to cross-react a human
  transcript probe
• with rat target mRNA
• Increase the stringency to avoid cross-reactivity
  of homologous
• probes

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Pros Cons

• Pros

• You can assay for highly homologous targets
  within one assay

• You can detect (and quantify) low-abundance
  transcripts

• You can work with the detection system that best
  suits
• your needs

• The assay is cost effective

• Cons

• One assay takes several days to complete (time
  for detection
• varies with probe label)

• The assay can be difficult to optimize

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Optimizing the Assay

• Use a control probe and control RNA

• Use in vitro transcription to label a molecular
• weight marker to run on your assay gel

• Vary the concentrations of your target RNA
• You should see a dose-response effect

• Vary the concentration of RNase
• You should see incomplete digestion of probe at
• very low concentrations

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TroubleshootingI Cant Detect My Target RNA!
WANT
GOT

• Maybe there is a problem with the orientation of
  the cDNA
• youre using for probe transcription

• Maybe your target RNA really isnt thereassay a
  tissue where
• it definitely will be found

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TroubleshootingThe Protected Fragment is Barely
There!
(1) Try adding more probe it may be the
limiting factor
WANT
GOT
(2) Try increasing the specific activity of your
probe
(3) Try using more target RNA
Maybe target really is low abundance!!
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TroubleshootingWhy Do I Have So Many Bands?
Increase the hybridization temperature
WANT
GOT
You may still see some non-specific bands, but
this is normal
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Similar Assays
The RPA gives results that are similar to many of
these assays. RPAs are more sensitive than many
of these and do not give many false
positives. They are often much less expensive,
too! RPAs are frequently used as a primary or
secondary screen to Detect differences. A
more rapid assay may be used after RPA
validation.

• S1 nuclease assay
• Subtractive hybridization
• Northern blot
• Quantitative RT-PCR
• TaqMan
• Microarray

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Examples from the Literature

• Differential effects of acute and chronic
  exercise on plasticity-related genes in the rat
  hippocampus revealed by microarray
• The neuropeptide Y receptors, Y1 and Y2, are
  transiently and differentially expressed in the
  developing cerebellum
• Down regulation of the IL-8 promoter by human
  papillomavirus type 16 E6 and E7 through effects
  on CREB binding protein/p300 and P/CAF