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To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.
Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.
Reverse-transcriptase polymerase chain reaction (PCR) has been the gold standard for mRNA quantitation. Two assays, digital PCR (dPCR)1 and quantitative, real time PCR (qPCR)2 are currently used. Of the two PCR techniques, dPCR has greater sensitivity than qPCR suggesting that it could be used to measure mRNA abundance in single cells. However, in our hands, dPCR analysis of low abundance mRNAs in pools of 5 to 10 oocytes per each experimental sample has produced data with low reproducibility and high variation3. This is likely due to the experimental error associated with RNA extraction and rever....
Animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Nebraska-Lincoln and all methods were performed in accordance with relevant guidelines and regulations. For this study, CD-1 outbred mice had ad libitum access to normal rodent chow and water; they were maintained in a 12:12 dark: light cycle.
1. Preparation of required media
Upon the completion of the protocol, the result will be individual images from confocal z-series (Figure 4A and Figure 5), stitched images (Figure 4C), and mRNA counts (Figure 4B). When multiplexing is performed, there will also be merged images showing the label for two different mRNAs (Fig.......
A series of minor steps during the protocol will ensure successful fluorescence and accurate counts of mRNAs. First, the protocol must be performed immediately after collection and fixation of the oocytes. Note that PVP is added to the 4% paraformaldehyde fixation buffer to prevent oocytes from sticking to each other. We found that it is necessary to perform the experiment immediately after the collection and fixation of the oocytes. Any delay results in a much lower fluorescence signal that would result in undercounting.......
We thank Dr. Daniel R. Larson for his generous help with the installation and use of the Spot Finding and Tracking Program 13 and the technical support of the University of Nebraska Lincoln Microscopy Core for the confocal microscopy imaging. This study represents a contribution of the University of Nebraska Agricultural Research Division, Lincoln, Nebraska and was supported by UNL Hatch Funds (NEB-26-206/Accession number -232435 and NEB-26-231/Accession number -1013511).
....Name | Company | Catalog Number | Comments |
(±)-α-Lipoic acid | Sigma-Aldrich | T1395 | Alpha Lipoic Acid |
Albumin, Bovine Serum, Low Fatty Acid | MP Biomedicals, LLC | 199899 | FAF BSA |
BD 10mL TB Syringe | Becton, Dickinson and Company | 309659 | 10 mL syringe |
BD PrecisionGlide Needle | Becton, Dickinson and Company | 305109 | 27 1/2 gauge needle |
Calcium chloride dihydrate | Sigma-Aldrich | C7902 | CaCl2-2H2O |
Citric acid | Sigma-Aldrich | C2404 | Citrate |
D-(+)-Glucose | Sigma-Aldrich | G6152 | Glucose |
Disodium phosphate | Na2HPO4 | ||
Easy Grip Petri Dish | Falcon Corning | 351008 | 35 mm dish |
Edetate Disodium | Avantor | 8994-01 | EDTA |
Extra Fine Bonn Scissors | Fine Science Tools | 14084-08 | Straight, Sharp/Sharp, non-serrated, 13mm cutting edge scissors |
Fetal Bovine Serum | Atlanta biologicals | S10250 | FBS |
Gentamicin Reagent Solution | gibco | 15710-064 | Gentamicin |
GlutaMAX-I (100X) | gibco | 35050-061 | Glutamax |
Gold Seal Micro Slides | Gold Seal | 3039 | 25 x 75mm slides |
Gonadotropin, From Pregnant Mares' Serum | Sigma | G4877 | eCG |
hCG recombinant | NHPP | AFP8456A | hCG |
Hyaluronidase, Type IV-S: From Bovine Testes | Sigma-Aldrich | H3884 | Hyaluronidase |
Jewelers Style Forceps | Integra | 17-305X | Forceps 4-3/8", Style 5F, Straight, Micro Fine Jaw |
L-(+)-Lactic Acid, free acid | MP Biomedicals, LLC | 190228 | L-Lactate |
Magnesium sulfate heptahydrate | Sigma-Aldrich | M2773 | MgSO4-7H2O |
MEM Nonessential Amino Acids | Corning | 25-025-Cl | NEAA |
Microscope Cover Glass | Fisher Scientific | 12-542-C | 25 x 25x 0.15 mm cover slips |
Mm-Nanog-O2-C2 RNAscope Probe | Advanced Cell Diagnostics | 501891-C2 | Nanog Probe |
Mm-Pou5f1-O1-C3 RNAscope Probe | Advanced Cell Diagnostics | 501611-C3 | Pou5f1 Probe |
MOPS | Sigma-Aldrich | M3183 | |
Paraformaldehyde | Sigma-Aldrich | P6148 | Paraformaldehyde |
PES 0.22 um Membrane -sterile | Millex-GP | SLGP033RS | 0.22 um filters |
Polyvinylpyrrolidone | Sigma-Aldrich | P0930 | PVP |
Potassium chloride | Sigma-Aldrich | 60128 | KCl |
Potassium phosphate monobasic | Sigma-Aldrich | 60218 | KH2PO4 |
Prolong Gold antifade reagent | invitrogen | P36934 | Antifade reagent without DAPI |
RNAscope DAPI | Advanced Cell Diagnostics | 320858 | DAPI |
RNAscope FL AMP 1 | Advanced Cell Diagnostics | 320852 | Amplifier 1 |
RNAscope FL AMP 2 | Advanced Cell Diagnostics | 320853 | Amplifier 2 |
RNAscope FL AMP 3 | Advanced Cell Diagnostics | 320854 | Amplifier 3 |
RNAscope FL AMP 4 ALT A | Advanced Cell Diagnostics | 320855 | Amplifier 4 ALT A |
RNAscope FL AMP 4 ALT B | Advanced Cell Diagnostics | 320856 | Amplifier 4 ALT B |
RNAscope FL AMP 4 ALT C | Advanced Cell Diagnostics | 320857 | Amplifier 4 ALT C |
RNAscope Fluorescent Multiplex Detection Reagents Kit | Advanced Cell Diagnostics | 320851 | FISH Reagent Kit |
RNAscope Probe 3-plex Negative Control Probe | Advanced Cell Diagnostics | 320871 | Negative Control |
RNAscope Probe 3-plex Positive Control | Advanced Cell Diagnostics | 320881 | Positive Control |
RNAscope Probe Diluent | Advanced Cell Diagnostics | 300041 | Probe Diluent |
RNAscope Protease III | Advanced Cell Diagnositics | 322337 | Protease III |
RNAscope Protease III & IV Reagent Kit | Advanced Cell Diagnostics | 322340 | FISH Protease Kit |
RNAscope Protease IV | Advanced Cell Diagnostics | 322336 | Protease IV |
S/S Needle with Luer Hub 30G | Component Supply Co. | NE-301PL-50 | blunt 30 gauge needle |
Sodium bicarbonate | Sigma-Aldrich | S6297 | NaHCO3 |
Sodium chloride | Sigma-Aldrich | S6191 | NaCl |
Sodium hydroxide | Sigma-Aldrich | 306576 | NaOH |
Sodium pyruvate, >= 99% | Sigma-Aldrich | P5280 | Pyruvate |
Solution 6 Well Dish | Agtechinc | D18 | 6 well dish |
Taurine | Sigma-Aldrich | T8691 | Taurine |
Tissue Culture Dish | Falcon Corning | 353002 | 60 mm dish |
Triton X-100 | Sigma-Aldrich | X100 | Triton X-100 |
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