Ribonucleic acid, or RNA, is a molecule that plays a central role in how genetic information from DNA is expressed within a cell. It acts as a messenger, carrying instructions from DNA to the cellular machinery that builds proteins. RNA molecules, however, are inherently unstable and prone to degradation by enzymes called RNases, making them challenging to work with directly in laboratory settings. To overcome this instability, scientists often convert RNA into a more stable form called complementary DNA, or cDNA. This DNA copy of an RNA molecule is then used for various molecular analyses. This article will explain the fundamental two-stage process of isolating RNA from biological samples and subsequently converting it into cDNA.
Isolating RNA from Biological Samples
Extracting RNA from biological samples involves carefully breaking open cells or tissues, a process known as lysis, while simultaneously deactivating RNase enzymes. These enzymes are abundant and can rapidly destroy RNA, necessitating their inactivation, often through the use of strong denaturants like guanidinium thiocyanate. This initial step ensures that all cellular contents, including RNA, are released into a solution, while also protecting the RNA from enzymatic breakdown.
Once cells are lysed, RNA must be separated from other macromolecules such as DNA and proteins. This separation can be achieved using two main strategies. Organic extraction methods involve a solution containing phenol and chloroform. When centrifuged, this mixture separates into three distinct layers: RNA remains in the upper aqueous phase, DNA collects at the interface, and denatured proteins reside in the lower organic phase.
Another widely used approach is solid-phase extraction, which employs silica-based spin columns. In this method, RNA selectively binds to a silica membrane in the presence of high salt concentrations. Contaminants are then washed away, and the purified RNA is subsequently released from the column using a low-salt solution. Following either extraction method, the isolated RNA is then precipitated, usually with alcohol, washed to remove residual contaminants, and finally dissolved in a suitable buffer for downstream applications.
Assessing RNA Purity and Integrity
Before proceeding to cDNA synthesis, evaluating the quality and quantity of the extracted RNA is a necessary step. UV spectrophotometry is a common method used to assess RNA purity and concentration. Nucleic acids absorb ultraviolet light strongly at 260 nanometers (nm), allowing for concentration determination, where an absorbance of 1.0 at 260 nm corresponds to approximately 40 micrograms per milliliter of RNA.
Purity is assessed by examining absorbance ratios. The A260/280 ratio indicates protein contamination, with an ideal ratio for pure RNA being around 2.0, while values below this may suggest protein or phenol contamination. The A260/230 ratio helps identify contamination from salts or organic solvents like guanidine isothiocyanate or phenol, with expected values ranging from 2.0 to 2.2. Lower values for either ratio suggest the presence of contaminants that could hinder subsequent molecular reactions.
Beyond purity, RNA integrity, or whether the molecules are intact or degraded, is equally important. This can be visually checked using gel electrophoresis, where intact total RNA from eukaryotic samples will show distinct 28S and 18S ribosomal RNA bands, ideally with the 28S band being about twice as intense as the 18S band. Degraded RNA, in contrast, appears as a smear rather than sharp bands, or shows a reduced 28S:18S ratio. More quantitative assessment is provided by automated microfluidic systems, which generate an RNA Integrity Number (RIN), ranging from 1 (totally degraded) to 10 (completely intact), with scores above 7 generally considered suitable for most downstream analyses.
The Process of Reverse Transcription
The conversion of RNA into cDNA is known as reverse transcription. This process creates a DNA copy from an RNA template, relying on a specific enzyme called reverse transcriptase. This enzyme reads the RNA sequence and synthesizes a complementary DNA strand. Reverse transcriptase was originally discovered in retroviruses.
The reverse transcription reaction requires several components. These include the RNA template, the reverse transcriptase enzyme, and deoxyribonucleotide triphosphates (dNTPs), which are the building blocks for the new DNA strand. A suitable buffer and an RNase inhibitor are also included to maintain enzyme activity and protect the RNA template. The choice of primer, a short DNA oligonucleotide that provides a starting point for the reverse transcriptase, is a significant experimental consideration.
Different priming strategies are employed depending on the RNA type and downstream application.
Oligo(dT) Primers
Oligo(dT) primers consist of a string of 12-18 deoxythymidines and bind specifically to the poly-A tail found on most eukaryotic messenger RNA (mRNA) molecules. These are often used for full-length cDNA cloning and constructing cDNA libraries from mRNA.
Random Hexamers
Random hexamers are short oligonucleotides, typically six nucleotides long, with random base sequences that bind non-specifically to all types of RNA, including ribosomal RNA and degraded RNA.
Gene-Specific Primers
Gene-specific primers are designed to anneal to a particular RNA sequence, allowing for the targeted synthesis of cDNA from a single transcript.
Key Applications of the Workflow
The stable cDNA molecules generated through reverse transcription serve as a versatile template for numerous molecular biology applications.
Quantitative Polymerase Chain Reaction (qPCR)
Quantitative Polymerase Chain Reaction, or qPCR, accurately measures the expression level of specific genes by monitoring DNA amplification in real-time using fluorescence. This technique allows researchers to quantify the amount of a particular RNA transcript present in a sample, providing insights into gene activity under different conditions.
RNA Sequencing (RNA-Seq)
RNA Sequencing, or RNA-Seq, is a high-throughput method that utilizes cDNA to analyze the entire collection of RNA molecules (the transcriptome) in a sample. By converting all RNA into cDNA, scientists can sequence millions of transcripts simultaneously, revealing comprehensive gene expression patterns, identifying novel RNA molecules, and detecting variations in RNA processing. This provides a detailed snapshot of gene activity within a cell or tissue at a given moment.
Gene Cloning and cDNA Libraries
cDNA is also instrumental in gene cloning and the construction of cDNA libraries. A cDNA library is a collection of cloned DNA sequences that represent the mRNA content of a specific cell or tissue, allowing researchers to study gene function and regulation. Unlike genomic DNA libraries, cDNA libraries contain only the coding sequences, making them particularly useful for expressing proteins in bacterial systems, as bacteria lack the machinery to process non-coding regions found in genomic DNA.