DNA and RNA extraction are the most crucial and basic method used in molecular biology and every genomics research. It is the starting point of any processes or product development and therefore, tailored extraction services are available with extraction service or combined with downstream analysis for:
DNA and RNA can be isolated from any biological material such as:
Human samples – blood, saliva, tissue, buccal swabs, and more
Any human and veterinary samples – cell cultures, lysates, blood, urine, feces, etcetera
Formalin fixed paraffin embedded (FFPE) samples
Samples for biobanking
Processed food samples, veterinary samples, microbiological cultures
There are two categories that are involved in purifying DNA and these are:
Isolation of chromosomal or genomic DNA constructs
Isolation of chromosomal or genomic DNA from prokaryotic or eukaryotic organisms.
Generally, the four important steps for successful nucleic acid purification are:
Effective disruption of cells or tissue
Degeneration of nucleoprotein complexes
Inactivation of nucleases
Keeping it away from contamination
The target nucleic acid should be free of contaminants as the quality and integrity of the isolated nucleic acid directly affects the results of all research after that. DNA is generally stable when there is appropriate storage conditions and can therefore, be prepared in batches or alternatively, whenever needed. The method of extraction depends on the:
Amount of sample available
Number of samples that needs to be extracted
Availability of equipment
The final use of the DNA
The target goal is to remove lipids, other proteins, and remove or degrade the RNA.
RNA is an unstable molecule with a very short half life once it is extracted from target tissues. Several naturally occurring RNA include:
Ribosomal RNA (rRNA)
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Special care and precautions are necessary in RNA isolation due to its susceptibility to degradation. It is unstable due to the presence of enzymes (RNases) present in blood and tissues. A good laboratory RNase free technique is needed for RNA extraction. The commonest methods for isolation are divided into two classes:
Utilization of guanidinium thiocyanate
Utilization of phenol and SDS
Type of Nucleic Acid Extraction
1. Conventional method
Guanidinium thiocyanate phenol chloroform extraction
Alkaline extraction method
Cetrylmethylammonium bromide (CTAB) extraction method
Ethidium bromide (EtBr) cesium chloride (CsCl) gradient centrifugation
Purification of poly (A)+ RNA by Oligp(dT) cellulose chromatography
2. Solid phase nucleic acid extraction
Samples should not be contaminated with other sources of DNA as any contaminant DNA will affect the data.
The most commonly used method to determine the quality is the ratio of absorbance at 260-280 nm.
Ratios around 1.8 means that it is good-quality, lower values indicate protein contamination, higher values indicate RNA contamination.
To determine size, DNAs can be run on a gel.
For an identity check of the DNA sample highly polymorphic microsatellite repeats can be determined and compared with another DNA source of the same sample. An example would be isolating DNA and comparing the alleles from both sources to see if it is from the same source.
For maximum stability, DNA is usually stored in a Tris-EDTA solution.
Short-term storage can be done at 4⁰C.
Long-term storage samples should be kept at -80⁰C.
Sealed vials can be used for storage as it provides minimal evaporation and therefore, maintains the necessary concentration while 96-well deep-well storage plates are used when there are larger numbers of samples.
The most important factors to remember for quality control of RNA are quantity, purity, and integrity.
The quality of RNA can be determined by the ratio of absorbance which should have idea values between 1.9-2.1. This can be done using a spectrophotometer.
Gel electrophoresis analysis should show bands of high molecular weight RNA that is between 7-15 kb in size, two predominant rRNA bands at approximately 5 kb (28s and 2 kb (18S) in a ratio of about 2:1, while the low molecular weight RNA is between 0.1-0.3 kb (tRNA, 5S). The integrity of the RNA can also be checked using a bioanalyzer.
Long tern storage is ideal at -80⁰C, or as an ethanol precipitate at -20⁰C.
One major concern for laboratories with large sample sizes are errors in sample identification and swapping.
Due to errors in identification and swapping, bar code labels should be used instead of hand labeling.
The use of robotics can also help minimize sample swapping.
DNA and RNA extraction has played important and crucial roles in helping researchers and scientists to manipulate molecular biology analysis to have a better understanding in the biology of the earth. Due to the rapid advancement of technology, DNA and RNA extraction has improved vastly however, weaknesses of the instruments should be bettered constantly by conducting quality control as it affects all subsequent results.
Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol. 2009; 2009: 574398.
Santella RM. Approaches to DNA/RNA extraction and whole genome amplification. Cancer Epidemiology, Biomarkers & Prevention. 2006; 15(9): 1585-1587.