Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. For base media (OMM), add 100 mM NaCl, 5 mM KCl, 0.5 mM KH₂PO₄, and 1.7 mM CaCl₂-2H₂O to 100 mL sterile water.
   NOTE: OMM medium can be stored for up to 1 month.
2. For complete media (....

Results

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.......

Discussion

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.......

Materials

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