Endy:RNase Protection Assay

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==Samples to be probed==  
==Samples to be probed==  
#Experimental Samples
#Experimental Samples
-
#*[[RNA Extraction|Total Cellular RNA]] ~10 μg total cellular RNA, would contain 10 fg –1 pg of rare RNA, ~300 pg of moderately abundant RNA.
+
#*[[RNA Extraction|Total Cellular RNA]] ~10 ?g total cellular RNA, would contain 10 fg �1 pg of rare RNA, ~300 pg of moderately abundant RNA.
#In Vitro Standard Curve Samples
#In Vitro Standard Curve Samples
-
#*[[RNA In Vitro Standards]] 1 fg 100 pg range suggested. ([[Molecular Cloning]]) 1 50 ng for high copy/plasmid-based, In carrier RNA to constant total mass, ~10 μg.
+
#*[[RNA In Vitro Standards]] 1 fg 100 pg range suggested. ([[Molecular Cloning]]) 1 50 ng for high copy/plasmid-based, In carrier RNA to constant total mass, ~10 ?g.
#Molecular Weight Standard
#Molecular Weight Standard
#* [[32P End-Labeled DNA Ladder]]
#* [[32P End-Labeled DNA Ladder]]
==Precipitation and Hybridization==
==Precipitation and Hybridization==
-
1) Combine:
+
#Combine:
-
- Sample or IVT standard RNA
+
#*Sample or IVT standard RNA
-
- Carrier RNA to equivalent total mass RNA. (Stock sol’n yeast tRNA = 1μg/μL)  
+
#* Carrier RNA to equivalent total mass RNA. (Stock sol�n yeast tRNA = 1?g/?L)  
-
- H2O to 99 μL total volume
+
#*H2O to 99 ?L total volume
-
- 10 μL (0.1volume) of 3M sodium acetate (pH 5.2)
+
#*10 ?L (0.1volume) of 3M sodium acetate (pH 5.2)
-
- 1 μL Riboprobe (suggested 2x105 to 10x105 cpm, 0.1-0.5 ng)
+
#*1 ?L Riboprobe (suggested 2x105 to 10x105 cpm, 0.1-0.5 ng) ''NOTE: may need significantly more to be in excess (cmc, 4-5-05)''
-
  *may need significantly more to be in excess (cmc, 4-5-05)  
+
#*250 ?L (2.5 volume) ice-cold 100% EtOH
-
- 250 μL (2.5 volume) ice-cold 100% EtOH
+
#Incubate 10 min at -80� C (''Optional, 20 minLonger incubation/spin yields better recovery of short or low quantity RNA.)''
-
2) Incubate 10 min at -80° C (Optional, 20 min**)
+
#Centrifuge 10 min at 4� C (''Optional, 25 min. Longer incubation/spin yields better recovery of short or low quantity RNA.)''
-
3) Centrifuge 10 min at C (Optional, 25 min**)
+
#Decant and wash pellet with 1mL of cold 80% EtOH  
-
** Longer incubation/spin yields better recovery of short or low quantity RNA.
+
#Decant. Incubate at room temp until all EtOH has evaporated, ~15 min.
-
4) Decant and wash pellet with 1mL of cold 80% EtOH  
+
#Dissolve Samples in 30 ?L hybridizaton buffer. Pipette numerous times to dissolve pellet. Roll bead of buffer across tube wall where RNA is deposited. (Heat to 60� C if difficult to resuspend RNA.)
-
5) Decant. Incubate at room temp until all EtOH has evaporated, ~15 min.
+
#Transfer samples to pcr tubes
-
6) Dissolve Samples in 30 μL hybridizaton buffer. Pipette numerous times to dissolve pellet. Roll bead of buffer across tube wall where RNA is deposited. (Heat to 60° C if difficult to resuspend RNA.)
+
#Run Hybridization Program on PCR block:
-
7) Transfer samples to pcr tubes
+
#*10 min at 85� C to denature RNAs
-
8) Run Hybridization Program on PCR block:
+
#*8-12 hours at annealing temp (specific to probe, usually 45-50� C, determine  optimal temp experimentally by hybridization with IVT at 25-65� C.)
-
10 min at 85° C to denature RNAs
+
#*Cool to 22.5� C (optional, cool to 4� C for 10-20 min. May stabilize hybrids prior to digest.)
-
8-12 hours at annealing temp (specific to probe, usually 45-50° C, determine  optimal temp experimentally by hybridization with IVT at 25-65° C.)
+
 
-
Cool to 22.C (optional, cool to C for 10-20 min. May stabilize hybrids prior to digest.)
+
==RNase Digest==
-
RNase Digest
+
#Prepare RNase digestion mixture. RNases must be added fresh just before digest.  
-
1) Prepare RNase digestion mixture. RNases must be added fresh just before digest.  
+
##For 1mL RNase digestion mixture, combine:
-
For 1mL RNase digestion mixture, combine:
+
##*994 ?L 1x buffer (300mM NaCl, 10mM Tris-Cl, pH 7.4, 5mM EDTA, pH 7.5)
-
  - 994 μL 1x buffer (300mM NaCl, 10mM Tris-Cl, pH 7.4, 5mM EDTA, pH 7.5)
+
##*4 ?L of RNase A (10 ?g/?L in 10mM Tris-Cl, 15mM NaCl)  
-
  - 4 μL of RNase A (10 μg/μL in 10mM Tris-Cl, 15mM NaCl)  
+
##*1 ?L RNase T1 (100 u/?L in 10mM Tris-Cl, 15mM NaCl)
-
  - 1 μL RNase T1 (100 u/μL in 10mM Tris-Cl, 15mM NaCl)
+
#Transfer samples to Eppendorf tubes.
-
2) Transfer samples to Eppendorf tubes.
+
#Add 300 uL RNase digestion mixture per sample. Incubate 60 min at 30� C. (Variable if signal to noise is poor after autoradiography.)
-
3) Add 300 uL RNase digestion mixture per sample. Incubate 60 min at 30° C. (Variable if signal to noise is poor after autoradiography.)
+
#Add 20 ?L of 10% SDS and 5 ?L of 20 mg/mL proteinase K to stop the reaction. Incubate 30 min at 37� C.  
-
4) Add 20 μL of 10% SDS and 5 μL of 20 mg/mL proteinase K to stop the reaction. Incubate 30 min at 37° C.  
+
 
-
EtOH Precipitation
+
==EtOH Precipitation==
-
1) Combine samples with  
+
#Combine samples with  
-
- 32 μL (0.1volume) of 3M sodium acetate (pH 5.2)
+
#*32 ?L (0.1volume) of 3M sodium acetate (pH 5.2)
-
- 800 μL (2.5 volume) ice-cold 100% EtOH
+
#*800 ?L (2.5 volume) ice-cold 100% EtOH
-
2) Incubate 10 min at -80° C (Optional, 20 min**)
+
#Incubate 10 min at -80� C (''Optional, 20 minLonger incubation/spin yields better recovery of short or low quantity RNA.)''
-
3) Centrifuge 10 min at C (Optional, 25 min**)
+
#Centrifuge 10 min at 4� C (''Optional, 25 min. Longer incubation/spin yields better recovery of short or low quantity RNA.)''
** Longer incubation/spin yields better recovery of short or low quantity RNA.
** Longer incubation/spin yields better recovery of short or low quantity RNA.
4) Decant and wash pellet with 1mL of cold 80% EtOH.  
4) Decant and wash pellet with 1mL of cold 80% EtOH.  
5) Decant. Incubate at room temp until all EtOH has evaporated, ~15 min.
5) Decant. Incubate at room temp until all EtOH has evaporated, ~15 min.
Gel Analysis
Gel Analysis
-
1) Add 10 μL RNA gel-loading buffer (with formamide) to samples and MW marker. (Use ~0.1 1 μL MW marker, depending on activity.)  
+
1) Add 10 ?L RNA gel-loading buffer (with formamide) to samples and MW marker. (Use ~0.1 1 ?L MW marker, depending on activity.)  
-
2) Incubate samples 5 min at 95° C. Transfer immediately to ice bath. Pulse-spin to consolidate sample.
+
2) Incubate samples 5 min at 95� C. Transfer immediately to ice bath. Pulse-spin to consolidate sample.
3) Load samples and markers in 6% polyacrylamide/8M urea gel, 0.75 mm thickness (See protocol, UreaAcrylGel_protocol) in 1x TBE buffer. See note on standard curves.
3) Load samples and markers in 6% polyacrylamide/8M urea gel, 0.75 mm thickness (See protocol, UreaAcrylGel_protocol) in 1x TBE buffer. See note on standard curves.
4) Electrophoresis: Run at 280 V for 7 cm mini-gel.  
4) Electrophoresis: Run at 280 V for 7 cm mini-gel.  
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7) Transfer gel to Whatman 3 MM filter paper that is 1 cm larger than the gel on all sides by laying paper on top of gel and inverting all.  
7) Transfer gel to Whatman 3 MM filter paper that is 1 cm larger than the gel on all sides by laying paper on top of gel and inverting all.  
8) Remove glass plate, place saran wrap over gel, do not wrap underneath.
8) Remove glass plate, place saran wrap over gel, do not wrap underneath.
-
9) Dry the gel on a pre-heated gel dryer for 30 min at 80° C.
+
9) Dry the gel on a pre-heated gel dryer for 30 min at 80� C.
Note: for higher percentage gels it is necessary to turn heat off prior to breaking the vacuum seal or gel will shatter.
Note: for higher percentage gels it is necessary to turn heat off prior to breaking the vacuum seal or gel will shatter.
10) While gel dries, erase Phosphorimager screen, 20 min on light table.
10) While gel dries, erase Phosphorimager screen, 20 min on light table.
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5) Repeat incubation overnight for higher signal intensity. Finish by erasing screen.
5) Repeat incubation overnight for higher signal intensity. Finish by erasing screen.
Image Analysis/ Quantitation
Image Analysis/ Quantitation
-
Use ImageQuant. (See protocol, ImageQuant.)
+
Use ImageQuant. (See protocol, ImageQuant.)
Additional Notes
Additional Notes
-
1) Standard Curves: It’s possible to condense samples from multiple standard curves in one lane, either by running samples of different sizes in the same lane, or by loading samples with different masses of standard in succession (if you do not care to see the MW of the standard.)
+
1) Standard Curves: It�s possible to condense samples from multiple standard curves in one lane, either by running samples of different sizes in the same lane, or by loading samples with different masses of standard in succession (if you do not care to see the MW of the standard.)
Buffers for Ribonuclease Protection Assay:
Buffers for Ribonuclease Protection Assay:
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pH to 6.4 w/ 1N HCl (~10 uL)
pH to 6.4 w/ 1N HCl (~10 uL)
adjust volume with nuclease-free water
adjust volume with nuclease-free water
-
store C wrapped in foil
+
store 4� C wrapped in foil
note: use disodium salt of PIPES (sesquisodium salt of PIPES worked fine also)
note: use disodium salt of PIPES (sesquisodium salt of PIPES worked fine also)
Line 115: Line 115:
For 10 mL, combine:
For 10 mL, combine:
-
- 50 μL of 3 M NaCl
+
- 50 ?L of 3 M NaCl
-
- 100 μL of 1 M Tris-Cl (pH 8.0)
+
- 100 ?L of 1 M Tris-Cl (pH 8.0)
- 9.85 mL nuclease-free H2O
- 9.85 mL nuclease-free H2O
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RNA Gel Loading Buffer with Formamide
RNA Gel Loading Buffer with Formamide
-
Store in the dark, at 4°C
+
Store in the dark, at 4�C
10 mL
10 mL
9.5 mL of deionized formamide
9.5 mL of deionized formamide
-
100 μL of 0.5 mM EDTA, pH 8.0
+
100 ?L of 0.5 mM EDTA, pH 8.0
-
2.5 μg of bromophenol blue
+
2.5 ?g of bromophenol blue
-
2.5 μg of xylene cyanol
+
2.5 ?g of xylene cyanol
-
2.5 μg SDS
+
2.5 ?g SDS
-
400 μL nuclease-free H2O
+
400 ?L nuclease-free H2O

Revision as of 14:07, 28 April 2005

RNase Protection Assay cmc

Contents

Samples to be probed

  1. Experimental Samples
    • Total Cellular RNA ~10 ?g total cellular RNA, would contain 10 fg �1 pg of rare RNA, ~300 pg of moderately abundant RNA.
  2. In Vitro Standard Curve Samples
  3. Molecular Weight Standard

Precipitation and Hybridization

  1. Combine:
    • Sample or IVT standard RNA
    • Carrier RNA to equivalent total mass RNA. (Stock sol�n yeast tRNA = 1?g/?L)
    • H2O to 99 ?L total volume
    • 10 ?L (0.1volume) of 3M sodium acetate (pH 5.2)
    • 1 ?L Riboprobe (suggested 2x105 to 10x105 cpm, 0.1-0.5 ng) NOTE: may need significantly more to be in excess (cmc, 4-5-05)
    • 250 ?L (2.5 volume) ice-cold 100% EtOH
  2. Incubate 10 min at -80� C (Optional, 20 minLonger incubation/spin yields better recovery of short or low quantity RNA.)
  3. Centrifuge 10 min at 4� C (Optional, 25 min. Longer incubation/spin yields better recovery of short or low quantity RNA.)
  4. Decant and wash pellet with 1mL of cold 80% EtOH
  5. Decant. Incubate at room temp until all EtOH has evaporated, ~15 min.
  6. Dissolve Samples in 30 ?L hybridizaton buffer. Pipette numerous times to dissolve pellet. Roll bead of buffer across tube wall where RNA is deposited. (Heat to 60� C if difficult to resuspend RNA.)
  7. Transfer samples to pcr tubes
  8. Run Hybridization Program on PCR block:
    • 10 min at 85� C to denature RNAs
    • 8-12 hours at annealing temp (specific to probe, usually 45-50� C, determine optimal temp experimentally by hybridization with IVT at 25-65� C.)
    • Cool to 22.5� C (optional, cool to 4� C for 10-20 min. May stabilize hybrids prior to digest.)

RNase Digest

  1. Prepare RNase digestion mixture. RNases must be added fresh just before digest.
    1. For 1mL RNase digestion mixture, combine:
      • 994 ?L 1x buffer (300mM NaCl, 10mM Tris-Cl, pH 7.4, 5mM EDTA, pH 7.5)
      • 4 ?L of RNase A (10 ?g/?L in 10mM Tris-Cl, 15mM NaCl)
      • 1 ?L RNase T1 (100 u/?L in 10mM Tris-Cl, 15mM NaCl)
  2. Transfer samples to Eppendorf tubes.
  3. Add 300 uL RNase digestion mixture per sample. Incubate 60 min at 30� C. (Variable if signal to noise is poor after autoradiography.)
  4. Add 20 ?L of 10% SDS and 5 ?L of 20 mg/mL proteinase K to stop the reaction. Incubate 30 min at 37� C.

EtOH Precipitation

  1. Combine samples with
    • 32 ?L (0.1volume) of 3M sodium acetate (pH 5.2)
    • 800 ?L (2.5 volume) ice-cold 100% EtOH
  2. Incubate 10 min at -80� C (Optional, 20 minLonger incubation/spin yields better recovery of short or low quantity RNA.)
  3. Centrifuge 10 min at 4� C (Optional, 25 min. Longer incubation/spin yields better recovery of short or low quantity RNA.)
    • Longer incubation/spin yields better recovery of short or low quantity RNA.

4) Decant and wash pellet with 1mL of cold 80% EtOH. 5) Decant. Incubate at room temp until all EtOH has evaporated, ~15 min. Gel Analysis 1) Add 10 ?L RNA gel-loading buffer (with formamide) to samples and MW marker. (Use ~0.1 � 1 ?L MW marker, depending on activity.) 2) Incubate samples 5 min at 95� C. Transfer immediately to ice bath. Pulse-spin to consolidate sample. 3) Load samples and markers in 6% polyacrylamide/8M urea gel, 0.75 mm thickness (See protocol, UreaAcrylGel_protocol) in 1x TBE buffer. See note on standard curves. 4) Electrophoresis: Run at 280 V for 7 cm mini-gel. Note: Glass plates should be warm to touch, usually 40-45 V/cm. dsRNA will run ~5-10% slower than DNA in this range; closer together at higher current. 5) Allow tracking dyes to run to appropriate distance. Note: In a 6% polyacrylamide gel: Xylene Cyanol runs at 110 nt, Bromophenol Blue runs at 25 nt Turn off power and dismantle electrophoresis set-up. Pry up one corner of the larger glass plate and cut one corner of the gel for orientation purposes. 6) Transfer glass plate with gel to flat bench top. Wisk off excess ddH20 by blotting edge with kimwipe. (Do not blot the gel surface.) 7) Transfer gel to Whatman 3 MM filter paper that is 1 cm larger than the gel on all sides by laying paper on top of gel and inverting all. 8) Remove glass plate, place saran wrap over gel, do not wrap underneath. 9) Dry the gel on a pre-heated gel dryer for 30 min at 80� C. Note: for higher percentage gels it is necessary to turn heat off prior to breaking the vacuum seal or gel will shatter. 10) While gel dries, erase Phosphorimager screen, 20 min on light table. 11) After removing gel from dryer, fold saran wrap around all. Autoradiography 1) Place dry gel in Phorphorimager cassette, facing erased screen. 2) Incubate 1 hour 3) Image screen on phosphorimager (settings: 176 micron pixel value, pmt = 650) 4) Erase screen 5) Repeat incubation overnight for higher signal intensity. Finish by erasing screen. Image Analysis/ Quantitation � Use ImageQuant. (See protocol, ImageQuant.)


Additional Notes 1) Standard Curves: It�s possible to condense samples from multiple standard curves in one lane, either by running samples of different sizes in the same lane, or by loading samples with different masses of standard in succession (if you do not care to see the MW of the standard.)

Buffers for Ribonuclease Protection Assay:

Hybridization Buffer (w/ Formamide); 5 mL

	from Molecular Cloning A1.12

200 uL 1M PIPES (40 mM) 10 uL 0.5M EDTA (1 mM) 400 uL 5M NaCl (0.4 M) 80% (v/v) deionized formamide: 4 mL

pH to 6.4 w/ 1N HCl (~10 uL) adjust volume with nuclease-free water store 4� C wrapped in foil note: use disodium salt of PIPES (sesquisodium salt of PIPES worked fine also)

RNase Digestion Mix Buffer

300 mM NaCl 10 mM Tris-Cl (pH 7.4) 5 mM EDTA (pH 7.5)

For 100 mL, combine: - 80 mL nuclease-free H2O - 10 mL of 3 M NaCl - 1 mL of 1 M Tris-Cl (pH 8.0) - 1 mL of 0.5 M EDTA pH to 7.5 with 1N HCl, if necessary. Adjust volume to 100 mL.

RNase Dilution Buffer

15 mM NaCl 10 mM Tris-Cl (pH 7.5)

For 10 mL, combine: - 50 ?L of 3 M NaCl - 100 ?L of 1 M Tris-Cl (pH 8.0) - 9.85 mL nuclease-free H2O


RNA Gel Loading Buffer with Formamide � Store in the dark, at 4�C 10 mL 9.5 mL of deionized formamide 100 ?L of 0.5 mM EDTA, pH 8.0 2.5 ?g of bromophenol blue 2.5 ?g of xylene cyanol 2.5 ?g SDS 400 ?L nuclease-free H2O

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