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

My Name is Elena Rand and I work at the Center for Medical Pathology at University of Copenhagen in Denmark. Here, approximately 60 people from various parts of the world are working with malaria. Our group is working with plasmodium foci, parasites.

They're the most violent ones causing malaria disease in humans plus mode. Foci room expresses surface proteins harboring various functions such as adhesion to host cells. The largest group of adhesion proteins are the so-called odum foci room erythrocyte membrane protein one, the so-called PfMP one.

There exists at least 60 variants of which only one has been believed to be transcribed for cell at a time during the blood stage of the infection. At our department, we believe that more than one protein may be transcribed at the same time in the parasites. One method to detect such activity is mRNA fluorescent in seed to hybridization fish.

Here we present a tailormade method of detection of mRNA, transcripts of two different genes in single nuclide of odum FCI infected erythrocytes. The method takes two days to complete. The first day involves preparation of RNA probes, adding them to the fixated parasites on glass slides and hybridization over nuts.

The second day involves antibody congregation, Fluor amplification, and visualization of the stained parasites in confocal microscope. To give an overview of the experiment, a flow chart of operations is shown critical Reagents And materials used in the experiment. RNAs zap dig labeling kit biotin, RNA, labeling mix, paraform aldehyde, glacial acetic acid, pepin anti dig antibody, anti biotin antibody, TSA PL plus fluorescence palate system, DPI RNAs, free micro tubes, ETH ethanol, wiped glass slides, autoclaved cover slips, sterile equipment such as scissors and forceps, a coupling glass jar.

The infected erythrocytes need to be checked by game sustaining to make sure that they are at the right developmental stage. IE the VAR transcribing ring stages in synchrony and in good condition. It is crucial to choose the exact developmental stage of the lifecycle when the transcription takes place.

Since individual RNA species are staged, specifically transcribed and rapidly degraded, the cells should be washed gently before making very high quality, thin blood films on clean glass slides, an even monolayer of infected red blood cells on the glass slide are then fixed in a 4%paraform aldehyde and 5%glacial acetic acid solution for 10 minutes. Then the glass slides are washed twice in PBS, remove the liquid from the slides gently with the tissue. Then perme alize the cells with a Prewarm 10 millimolar hydrogen chloride solution before adding the Pepsi in.

Incubate this at 37 degrees centigrade for two minutes prior to the hybridization. The probes are denatured at 65 degrees centigrade for five minutes, rapidly chill ice, and then added to the cells, which are now fixed On the glass slide, Put a cover slip on top of the cells and remove any air bubbles with a small pipet tip. Humidify the hybridization chamber by adding drops of double distilled water at the periphery of the chamber so the slides remain humid in the chamber.

During the hybridization procedure, put the glass slides into the chamber hybridize overnight at 48 degrees centigrade. After 16 to 17 hours of hybridization, the slides are carefully removed from the hybridization chamber. From this step onwards, there is no absolute requirement for an RNAs free environment.

Wash the slides in a glass jar filled with TNT buffer, making sure that the cover slips come off from the glass slide. Repeat the washing procedure three times, then block them in TNB buffer for half an hour. Add the HRP conjugated anti dig, or anti biotin labeled antibodies to the cells.

Both types of labeled antibodies can be added simultaneously. If the experiment aims to detect two mRNA transcripts in the cells, the antibodies should be pre-diluted. In TNB buffer, incubate for one to two hours, wash three times in TNT buffer, then add one fluorochrome to the washed cells, A diluted FSE or cyan.

Three solutions need to be freshly prepared. From this step onwards, it's important not to expose the slides directly to strong light. Incubate the glass slides for 10 to 15 minutes with the fluorochrome.

If detecting two mRNA transcripts after the first incubation, wash out the first dye and add the second. Make a final wash in TNT Buffer. Immerse the slides in double distilled water before air drying them.

Add the dappy and put a cover slip on top of the slide. Remove any air bubbles and seal the edges of the cover slip with quick drying nail varnish or polish. Here we see a single infected erythrocyte containing one parasite.

The blue, the DPE represents the DNA of the parasite, and the green represents the mRNA of one gene being transcribed. In the other image, the red color represents the other gene's mRNA, and in the third image we can see the dual stain of both gene transcription. The images also reveal that the mRNAs attached to are superimposed on the nuclear DNA and not generally lying on top or beside it.

In 99%of the infected erythrocytes, we can detect the mRNA of two genes being transcribed. An important aspect of this experiment is to check that the probes are not annealing to other genes. In this image, we have incubated the infected erythrocytes with both probes, but only one probe is a kneeling IE.The other genes are not being transcribed here.

This situation is reversed on these other images in the visualized infected erythrocytes. The odium phos palm parasites have generally well-preserved membranes with well-defined staining of both DNA and RNA. If the cells are for some reason of poor quality, staining will also become poor.

As is shown in this image, the DNA and the RNA will appear diffuse, cloudy, and relatively undefined. To conclude, it can be said that this method can be easily applied to other bloodborne parasites. Our transcription data have also been supported by experiments to detect the translated proteins from the analyzed mRNAs using specific antibodies targeting the surface expressed pf EMP one proteins on infected cells.