Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues (v2)

Chi-Chih Wu

May 30, 2024

Version 2

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues (v2) V.2

DOI

dx.doi.org/10.17504/protocols.io.q26g714e8gwz/v2

Chi-Chih Wu¹

¹Biodiversity Research Center, Academia Sinica, Taipei 11529, Taiwan

Chi-Chih Wu

IPMB

DOI: dx.doi.org/10.17504/protocols.io.q26g714e8gwz/v2

Protocol Citation: Chi-Chih Wu 2024. Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues (v2). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g714e8gwz/v2Version created by Chi-Chih Wu

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: May 10, 2024

Last Modified: May 30, 2024

Protocol Integer ID: 100911

Abstract

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Ｗe develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. 

Section permeabilization
 
A
1. The slides with
  sections are taken out the freezer and 
  equilibrated to RT for 40 mins.
2. Permeabilized in 20 ug/ml proteinase k
  for proper duration
3. Quickly wash in DEPC-PBS
4. Quickly dehydrate the slides in EtOH
  (50, 70, 99 %) and then air dry 
5. Mount the secure seal reaction
  chambers onto the slides.
6. Add 1x DEPC-PBS-tween 0.05 % (Wash
  buffer) into the chambers to keep the slides wet until RT reaction ready
 

Mixture for miRNA hybridization (50 ul)
 
ABC
 StockFinal
Formamide100%50%
SSC20x%5x
tRNA10 mg/ml0.5 ug/ul
Denhardt's50x1x
LNA probe A10 uM2-3 pmole
DEPC-H2O- 
1.  process
A
Hybridization below the predicted melting temperature of the probe, about 2 C, for an hour
Wash with 0.1X SSC three times at the temperature set in the Step 1
Wash with 2X SSC at RT once
Wahs wtih the wash buffer (PBS, 0.05% Tween-20) once
 

 

Mixture for mRNA cDNA synthesis (50 ul)
 
ReagentStockFinal
NEB Tag DNA ligase40U/ul0.5 U/ul
Rnase H5 U/ul0.4 U/ul
Ribolock Rnase inhibitor40 U/ul1 U/ul
NEB Tag ligase buffer10x1x
BSA20 ug/ul0.2 ug/ul
KCl1 M0.05 M
Formamide100%20%
Pd_A10 uM0.1 uM
Pd_B10 uM0.1 uM
Pd_C10 uM0.1 uM
DEPC-H2O  
 Process
1. Add ligation
  mixture in chambers, seal with adhesive film
2. Incubate for 30
  min at 37 C followed by 45 min at 48 C
3. Wash 2x, 1x DEPC-PBS-Tween 20, 0.05%
 
 

Mixture for miRNA and mRNA padlock probe hybridization and ligation (50 ul)
 
ReagentStockFinal
NEB Tag DNA ligase40U/ul0.5 U/ul
Rnase H5 U/ul0.4 U/ul
Ribolock Rnase inhibitor40 U/ul1 U/ul
NEB Tag ligase buffer10x1x
BSA20 ug/ul0.2 ug/ul
KCl1 M0.05 M
Formamide100%20%
Pd_A10 uM0.1 uM
Pd_B10 uM0.1 uM
Pd_C10 uM0.1 uM
DEPC-H2O  
process
 
A
1. Add ligation
  mixture in chambers, seal with adhesive film
2. Incubate for 30
  min at 37 C followed by 45 min at 48 C
3. Wash 2x, 1x DEPC-PBS-Tween 20, 0.05%
 
 

Rolling circle amplification (50 ul)
 
ReagentStockFinal
Phi 29 polymerase10 U/ul1 U/ul
Ribolock Rnase inhibitor40 U/ul1 U/ul
10 X phi 29 buffer10 x1 x
dNTP10 mM0.25 mM
BSA20 ug/ul0.2 ug/ul
Glycerol50%5%
DEPC-H2O  
process
 
A
1. Add reaction
  mixture and seal chamber
2. Incubate for over night at 37 C
3. Wash 2x, DEPC-PBS-Tween 20, 0.05 %
 
 

Mixture for detection oligo hybridization (50 ul)
 
ABC
ReagentStockFinal
Hyb mixture4 x2 x
Detection oligo 1-FITC1 uM0.1 uM
Detection oligo 2-Cy31 uM0.1 uM
Detection oligo 3-Cy51 uM0.1 uM
DEPC-H2O  
process
 
1. Add reaction
  mixture
2. Incubate for 30 min at 37 C
3. Wash 2x, DEPC-PBS-Tween 20, 0.05 %
4. Dehydrated by EtOH 50, 70, 99 %; then
  air dry.
5. Mount cover slips
 
 

	A
	1. The slides with sections are taken out the freezer and equilibrated to RT for 40 mins.
	2. Permeabilized in 20 ug/ml proteinase k for proper duration
	3. Quickly wash in DEPC-PBS
	4. Quickly dehydrate the slides in EtOH (50, 70, 99 %) and then air dry
	5. Mount the secure seal reaction chambers onto the slides.
	6. Add 1x DEPC-PBS-tween 0.05 % (Wash buffer) into the chambers to keep the slides wet until RT reaction ready

A	B	C
	Stock	Final
Formamide	100%	50%
SSC	20x%	5x
tRNA	10 mg/ml	0.5 ug/ul
Denhardt's	50x	1x
LNA probe A	10 uM	2-3 pmole
DEPC-H2O	-

	A
	Hybridization below the predicted melting temperature of the probe, about 2 C, for an hour
	Wash with 0.1X SSC three times at the temperature set in the Step 1
	Wash with 2X SSC at RT once
	Wahs wtih the wash buffer (PBS, 0.05% Tween-20) once


Reagent	Stock	Final
NEB Tag DNA ligase	40U/ul	0.5 U/ul
Rnase H	5 U/ul	0.4 U/ul
Ribolock Rnase inhibitor	40 U/ul	1 U/ul
NEB Tag ligase buffer	10x	1x
BSA	20 ug/ul	0.2 ug/ul
KCl	1 M	0.05 M
Formamide	100%	20%
Pd_A	10 uM	0.1 uM
Pd_B	10 uM	0.1 uM
Pd_C	10 uM	0.1 uM

DEPC-H2O


	1. Add ligation mixture in chambers, seal with adhesive film
	2. Incubate for 30 min at 37 C followed by 45 min at 48 C
	3. Wash 2x, 1x DEPC-PBS-Tween 20, 0.05%

	A
	1. Add reaction mixture and seal chamber
	2. Incubate for over night at 37 C
	3. Wash 2x, DEPC-PBS-Tween 20, 0.05 %


	1. Add reaction mixture
	2. Incubate for 30 min at 37 C
	3. Wash 2x, DEPC-PBS-Tween 20, 0.05 %
	4. Dehydrated by EtOH 50, 70, 99 %; then air dry.
	5. Mount cover slips

Public workspaceSimultaneous detection of miRNA and mRNA at the single-cell level in plant tissues (v2) V.2

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues (v2) V.2