Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues (v2)
Chi-Chih Wu
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.
Steps
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
| 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 |
- process Hybridization temp. Wash w/ 0.1X SSC at 37C Wash w/ 2X SSC at RT, x1 Wahs w/ wash buffer (PBS, 0.05% Tween-20), x1
Mixture for mRNA cDNA synthesis
| A | B | C |
|---|---|---|
| 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 |
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
| A | B | C |
|---|---|---|
| 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 |
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%
Rolling circle amplification
| A | B | C |
|---|---|---|
| Reagent | Stock | Final |
| Phi 29 polymerase | 10 U/ul | 1 U/ul |
| Ribolock Rnase inhibitor | 40 U/ul | 1 U/ul |
| 10 X phi 29 buffer | 10 x | 1 x |
| dNTP | 10 mM | 0.25 mM |
| BSA | 20 ug/ul | 0.2 ug/ul |
| Glycerol | 50% | 5% |
| DEPC-H2O |
process 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
| A | B | C |
|---|---|---|
| Reagent | Stock | Final |
| Hyb mixture | 4 x | 2 x |
| Detection oligo 1-FITC | 1 uM | 0.1 uM |
| Detection oligo 2-Cy3 | 1 uM | 0.1 uM |
| Detection oligo 3-Cy5 | 1 uM | 0.1 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
