RNA-Seq is a process that uses high-throughput sequencing to determine the presence and quantity of RNA in a biological sample at a given moment. RNA-seq is a common technique used to study the cell’s transcriptome to analyze what genes are expressed under a given circumstance. RNA-Seq facilitates the study of alternatively spliced gene transcripts, post-transcriptional modifications, gene fusions, mutations/SNPs, and changes in gene expression over time or in different groups/treatments.
In the first step of the sample preparation process RNA is isolated from a given cell environment. This can be done using spin-column kits, magnetic bead kits or fully automated extractors. These kits contain buffers for lysing cells and stabilizing RNA against degradation, enzymes that disable RNases, and columns that collect the RNA. Samples must also be treated with DNase, which is not included in most kits, to reduce the amount of genomic DNA present in the sample.
Following extraction, the preferred RNA type is separated from the undesirable RNA by enrichment of specific sequences. If working with a eukaryotic transcriptome, the messenger RNA (mRNA) and non-coding transcripts are separated from ribosomal (rRNA) by Poly(A) selection. Poly(A) selection is performed by mixing the eukaryotic RNA with poly(T) oligomers covalently attached to magnetic beads which bind RNA transcripts with a 3’ poly(A) tail and eliminate other types of RNA.
RNA enrichment ensures the signal for preferred RNA sequences will be magnified relative to other RNA sequences in the sample. The selected RNA sequences are then transcribed to complementary DNA (cDNA) by reverse transcriptase. DNA is more stable than RNA and can be amplified by PCR, making cDNA synthesis an essential step in the sample preparation process.
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A sequencing library can be prepared by fragmenting the cDNA with enzymes, sonication, or nebulizers. Then the nucleic acid fragments would be separated based on size. Micro-RNAs are then filtered out and can be analyzed separately as they are too small for amplification.
Next step is clonal amplification. This step amplifies the cDNA library in preparation for high-throughput sequencing using Next Generation Sequencing (NGS) techniques such as emulsion PCR and bridge PCR. Read our comprehensive guide on Next-Generation Sequencing for more information on these techniques. At this stage, the sequencing adapters and barcodes are added to the library in preparation for sequencing. It is also beneficial to perform quantification and analysis of the libraries prior to loading onto the sequencer to ensure high quality results.
Sequencing may be done on different platforms. Illumina, Nanopore, and Ion Torrent present different technologies and workflows for high-throughput sequencing depending on your needs. Details on these methods can be found on the Next-Generation Sequencing page. A sequencing run generates short reads (50-500 bp) that need to be processed and analyzed to gain information from the sequencing run.
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After the short reads are obtained, a bioinformatics approach enables reconstruction of the transcriptome RNA sequences. Primary analysis may be done using available bioinformatics tools. Read sequences and associated information, such as read quality scores, are stored in a FASTQ file format. FASTQ files are filtered based on the quality scores and trimmed to remove adapter sequences.
The processed reads can be stitched together based on sequence overlap to reconstruct longer length reads, referred to as contigs. The contigs are then mapped to a reference genome and used to quantify the abundance of transcripts for a given gene. Reads may also be aligned directly to a reference genome, using spliced aligners capable of aligning reads that span exon-exon junctions. Alignment is typically done by software tools like STAR, HISTAT or TopHat among many others.
Secondary analysis is performed to identify differentially expressed genes or RNA variants, such as those associated with differently spliced mRNA. Tools like Sailfish, RSEM, and BitSeq will quantify gene expression levels, while MISOand SpliceSEQ quantify alternatively spliced genes. Many bioinformatics software tools are available for public use and there are extensive resources like libraries, reviews, and roundups to aid in the search to find the best tool available for a specific application.
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1. Developing a primary culture
Primary culture refers to the cells that are isolated directly from the tissue of interest and proliferated until they reach confluence, or occupy all the available substrate. One method of acquiring cells for primary culture entails sampling from the tissue directly. Cells taken in this manner must be disaggregated using enzymatic or mechanical means before they are placed on the substrate.
After the primary culture reaches confluence, the cells have to be subcultured by transferring them to fresh growth medium. At this stage the cell culture is no longer considered primary and becomes secondary culture, or cell line. Cell lines derived from primary cultures are finite, which means they have a limited number of cell divisions that is a genetically determined. The loss of ability to proliferate is called senescence.
Another way of acquiring cell culture is by using an established cell strain. These are continuous, or immortalized, cell lines that have been mutated and lost the ability to undergo senescence. These cells can continuously divide and are optimal cell lines for prolonged studies. The most common immortalized cell lines used for primary culture include HeLa cells and HEK 293 cells, among others
2. Developing Cell Lines
Regardless of the cell line type, it is necessary to propagate the cell line by continuously passaging the cells into fresh media, a process known as splitting the cells. To split cells, cell media is aspirated via a vacuum and the cells are washed in warmed Phosphate Buffered Saline (PBS) to remove residual media. If the cells are anchorage-dependent and rely on adherence to a surface, the cells can be treated with trypsin, a chemical that causes cells to temporarily lose their adherence. Trypsin, however, is toxic and should not be applied for long.
Trypsin toxicity is neutralized by adding growth media. For mammalian cells, this growth media is typically DMEM supplemented with Fetal Bovine Serum (FBS) and antibiotics. From this solution, the media and cells can be partitioned, or split, into new cell culture plates. A common practice is to always maintain at least one plate for further splitting and use the rest to split into additional plates that will be used for experiments.
While the above example is for anchorage-dependent mammalian cells, a similar principle applies to other types of cells including those in suspension and spore colony culture. In suspension culture, cells from a developed culture may be split by transfer into fresh growth media. In colony culture, spores or colonies may be taken from a developed plate and streaked across fresh media plates
Different cells require different growth conditions. While conditions may vary from cell type to cell type, most cells are grown in incubators to maintain optimum growth conditions. Incubators have the ability to maintain an optimum temperature (37oC for most cells) and precise O2 and CO2 levels. Additionally, cells will require a growth medium that is unique to each cell type. Media contains the nutrients, hormones, and buffers for cells to grow and should be continually changed. The right media and incubation conditions are critical for cell growth.
3. Maintaining Cell Lines
Successful cell culture depends on keeping the cells free from contamination by microorganisms such as bacterial, fungi, and viruses. Nonsterile supplies, media, reagents, airborne particles, unclean incubators, and dirty work surfaces are all sources of biological contamination.
Aseptic technique is the best barrier to prevent contamination by invasive microorganisms and should always be maintained. Aseptic technique includes:
- Only opening cell culture in a sterile environment such as a cell culture hood with working airflow.
- Sterilizing the work area before and after each handling of the cells with proper cleaning reagents. 70% ethanol is the most common choice.
- Using gloves, lab coats, and other PPE as needed to prevent contamination of the samples and maintain personal safety.
- All media and solutions should be opened and/or mixed within a cell culture hood.
- Using only sterile glassware and disposable pipettes. Dispose of pipettes after each use.
- Opening media and other containers only when ready to use.
However, even with proper aseptic technique, contamination can happen. In these cases, the incubator and cell culture hood should be thoroughly cleaned to prevent other samples from being contaminated. If contaminated cells or media are critical to work, they may be treated with antibiotics to kill the contaminants - though care should be taken as antibiotics may cross-react with the cells of interest. In most cases, the contaminated cells and media should be thrown out and prepared anew.
Long-term storage of cells is a useful way to backup any experiments relying on a certain subculture or strain of cells. The most common method is Cryopreservation. Cryopreservation requires a surplus of cells be taken from cell culture and mixed with a protective agent, typically DMSO or glycerol, before being stored below –130°C.