What do you know about micromanipulation?
Micromanipulation is a method where individual cells are extracted manually from the sample. The pros, in this case, are that it guarantees that each cell captured is captured successfully, while the cons are that it is extremely slow and low throughput. Microdissection, on the other hand, is used for extracting single cells from tissues. Microdissection utilizes a laser to take individual cells from said tissue and attach them to a ‘thin film that can then be removed.’ FACS, or fluorescence-activated cell sorting, is a type of flow cytometry. It sorts a heterogeneous cell population into containers one cell at a time, using the fluorescence to determine where it should be sorted. This is a much quicker way to separate and sort cells, but to use FACS, the tissue sample must be dissolved with enzyme treatment, which has the chance of injuring the cells and altering results. Microfluidics employs integrated fluidic circuits in order to isolate individual cells. Like FACS and microdroplets, this is a very fast method, but it has quite a few limitations. It requires that all of the cells are around the same size, and it can miss capturing cells that are not spherical or are sticky, and it requires a relatively large input size (~1000 cells).
Following single cell capture, the cells must then all be lysed (their cell membranes are broken down) so that the RNA inside is accessible for the following steps. Then comes reverse transcription, which can be accomplished in two ways: polyA tailing + second strand synthesis or template switching. PolyA tailing + second strand synthesis is the historical method of transcription, and it involves having an anchor sequence followed by a polyT tail. This is complementary to the polyA tail at the 3’ end of mRNA molecules and allows the primer to bind to the mRNA molecules. This polyA tailing is a vital step to both methods of reverse transcription. Then, the first strand of cDNA is synthesized via reverse transcriptase-based off of the mRNA molecule template. Once the mRNA degrades, DNA polymerase synthesizes the second strand of cDNA from the 5’ to the 3’ end. The con to this way of second strand synthesis, however, is that it often underrepresents the 5’ ends of cDNA. Template switching, on the other hand, employs a chimeric DNA/RNA template switching oligonucleotide and the Moloney murine leukemia virus (MMLV) reverse transcriptase.