Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Summary

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes ¹. The technique is adaptable and can be used on different types of genes, cells and organisms.

Abstract

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.

The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE¹.

Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System² (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Protocol

1. Generation of Freshly Selected Infected Erythrocytes

For this assay, the best results are obtained when using freshly selected cultures for surface expression of PfEMP1 protein. In this particular experiment the 3D7 P. falciparum lineage was selected using specific antibodies as previously described¹.

Day 1

1. Harvest 200 μl of packed blood cells from a P. falciparum culture containing 2-5 % late stage IE by centrifugation at 800 x g for 8 min at room temperature.
2. Re-suspend the blood pellet in 2 ml of 37 °C warm 0.75 % gelatin solution in a 14 ml sterile ....

Results

Figure 1 illustrates a flowchart of the major steps and the time duration of the RNA FISH methodology.

A series of representative images of well and poorly preserved stained mRNA FISH experiments using single P. falciparum IE are shown in Figure 2. This intracellular protozoan has a small (1-1.5 μm diameter) nucleus which is stained blue with DAPI. Twenty four hours after erythrocyte invasion in P. falciparum the process of schizogony, i........

Discussion

RNA FISH analysis, in contrast to methods such as Northern blotting and RT-PCR, allows discrimination of specific mRNA transcripts at the single cell level. This makes it possible to discriminate between transcriptionally active and inactive cells, in this example, P. falciparum parasitic protozoa inside human red blood cells. Such whole-cell observations are often necessary and may unravel important and novel transcriptional patterns¹.

Although other RNA FISH methods have.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic Acid	Sigma/Aldrich	338826-100ml
Albumax media: RPMI 1640 Glutamine solution	Lonza	BE12-115F	500 ml RPMI 1640 5 ml glutamine solution
Albumax media: Gentamycin sulphate Albumax-solution	Lonza	BE02-012E	2.5 ml gentamycin sulphate 50 ml Albumax-solution
Albumax-solution: Hypoxanthine	Sigma-Aldrich	H9377	0.8 g Hypoxanthine
Albumax-solution: AlbuMAX II	Invitrogen	11021-037	200 g Albumax
Albumax-solution: RPMI 1640	Lonza	BE12-115F	4 liter RPMI 1640 Dissolve with magnet at max. 50 °C. Filter sterilize and store at -20 °C in aliquots.
Amberlite	Sigma	A5710-110G	Resin to deionize formamide.
Anti-biotin goat pAb Peroxidase Conjugate	Calbiochem	203206
Anti-Dig antibody	Novus biologicals	NB100-41330
Anti-fade reagent with DAPI	Invitrogen	P36931	The mounting media has to cure for 24 hr before sealing the slide completely.
Biotin RNA Labeling Mix	Roche	11685597910
Camera digital	Nikon digital sight DC-F11
Coverslips	Menzel-glaser	631-1570
culture flask 25 cm2 Nunclon surface	Nunc	156340
DEPC	Fluka/Sigma	32490-100ml	1 ml DEPC in 1000 ml deinonized water. Add a stir bar and stir for 12 hr. Autoclave for 30 min.
Digital camera	Nikon digital sight DC-F11
Dynabeads Protein A	Invitrogen	10002D
DynaMaq-15 Magnet	Invitrogen	123-01D
Formamide bioultra 99 %	Fluka/Sigma	47671-1l-F	Deionized formamide: 5 g of ion exchange resin per 100 ml of formamide. Stir 30 min. Filter through Whatman paper.
Gelatine 0.75% solution: Gelatine	Sigma-Aldrich	G2500	3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 °C to dissolve. Filter sterilize when 56 °C. Store at -20 °C in aliquots.
Gelatine 0.75% solution: RPMI 1640	Lonza	BE12-115F	3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 °C to dissolve. Filter sterilize when 56 °C. Store at -20 °C in aliquots.
Glutamine solution: L-glutamin	Sigma-Aldrich	G3126	14.6 g L glutamine in 500 ml 0.9 % NaCl. Dissolve, filter sterilize and store at -20 °C in aliquots.
Glutamine solution: HCl	Sigma	H1758	14.6 g L glutamine in 500 ml 0.9 % NaCl. Dissolve, filter sterilize and store at -20 °C in aliquots.
Hybridization solution: Formamide Bio ultra 99% SSC 20x	Fluka/Sigma	47671-1l-F	Total 20 ml, keep frozen at -20 °C in aliquots 10 ml deionized formamide
Hybridization solution: Denhardt's 50x concentrate	Sigma	D2532	5 ml 20xSSC
Hybridization solution: Yeast tRNARoche blocking reagent	Sigma	R-6750	2 ml 50x Denhardt's 250 μl 20 mg per ml yeast tRNA
Hybridization solution: Salmon sperm DNA	Fluka/Sigma	31149-106GF	0.4 g Roche blocking reagent 1 ml of 10 mg per ml salmon sperm DNA (Critical: denature salmon spermDNA at 96 °C for 5 min before adding to the hybridization solution)
Hybridizer ThermoStar 100 HC4	Quantifoil Instruments GmbH	1004-0011	Can be replaced by hybridization oven and RNase free hybridization chambers padded with DEPC water.
Immersion oil UV transparent fluorescence free	Sigma	10976-1EA
Immunofluorescence or confocal microscope	Nikon D-Eclipse TE2000C
Nailpolish	Available in any drugstore
PBS 20x:NaCl KCl Na2HPO4 x 2H2O KH2PO4 DEPC deionized H2O			Ajust pH to 7.4 160 g 4 g 23 g 4 g 1 l
Paraformaldehyde 4 %	Fluka/chemika	76240	4 g PFA in 80 ml PBS/DEPC. Heat to 65 °C until the PFA dissolves. Add 20 ml PBS, allow the solution to cool. Adjust the pH to 7.4. Filter. Store in aliquots at -20 °C.
Paraformaldehyde 4 %/ Acid acetic 5 %			950 μl paraformaldhyde + 50 μl Acetic acid
Pepsin	Sigma/Aldrich	P7000
Peroxidase-conjugated anti-biotin	Calbiochem	203206
RBC-wash media: RPMI 1640 Glutamine solution	Lonza	BE12-115F	500 ml RPMI 1640 5 ml glutamine solution
RBC-wash media: Gentamycin sulfate	Lonza	BE02-012E	2.5 ml gentamycin sulphate
RNase	Sigma/Aldrich		Make a 10 mg/ml stock solution.
RNase free 1.5 ml tube	Ambion	AM12450
RNase Zap	Ambion	AM9780
Slides 4 wells 11 mm	Thermo Scientific	MENZXER306W
SSC 20x: NaCl	Sigma/Aldrich	S9625	3 M NaCl (175 g/l) 0.3M Na3 citrate x H2O (88 g/l) Adjust to pH 7.0 with 1M HCl
SSC 20x: Sodium citrate dehydrate	Sigma/aldrich	W302600	3 M NaCl (175 g/l) 0.3M Na3 citrate x H2O (88 g/l) Adjust to pH 7.0 with 1M HCl
SSPE 20x: NaCl	Sigma/Aldrich	S9625	175.3 g NaCl
SSPE 20x: NaH2PO4	Sigma/Aldrich	S0751	27.6 g NaH2PO4.
SSPE 20x:4 EDTA powder			9.4 g EDTA powder Add DEPC water, adjust pH 7.4. Autoclave for 20 min
TNB buffer: TNT buffer			10 ml TNT buffer
TNB buffer: Blocking reagent			0.05 g Blocking reagent To dissolve the blocking reagent, heat the solution to 60 °C for one hour with stirring. Store at -20 °C. (Derived from the Perkin Elmer TSA-protocol.)
TNT buffer: Tris/HCl	Sigma	T1503	1M Tris/HCl, pH 8.0
TNT buffer: NaCl	Sigma Aldrich	S9625	100 ml
TNT buffer: Tween20	Sigma	93773	5 M NaCl 30 ml 1 ml DEPC dH2O 869 Ml Adjust pH to 7.5 at room temperature. (Derived from the Perkin Elmer TSA-protocol.)
TSA plus Cyanine3/ Fluorescein system	Perkin Elmer	NEL753000IKT	Read the protocol from the TSA Plus Fluorescence Palette System carefully before starting the experiment.
Tubes 14 ml sterile	Almeco - CM LAB Aps	91016