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How to Test for Nucleic Acids

Testing for nucleic acids is carried out in medical diagnostics in order to detect pathogens, such as viruses or bacteria. These tests are much more rapid and, therefore, enable a physician to start treatment for a patient who ordinarily would have had to wait for confirmation of infection using more lengthy and tedious antibody-based assays. The process is also known as a nucleic acid amplification test, and includes a method known as "reverse-transcriptase amplification" by polymerase chain reaction. Since this is a highly specialized scientific procedure, advanced knowledge and expertise in molecular biology techniques and theory is needed.

Things You'll Need

  • PCR tubes
  • PCR cycler
  • Pipettes and filter tips
  • Gloves
  • Cells
  • Trizol or other RNA extraction reagent
  • PCR buffer
  • Taq polymerase
  • RT-PCR primers at 1 mg/ml concentration
  • RNase-free water
  • dNTPs
  • MgCl2
  • Random primers at 1.8 mg/ml concentration
  • SuperScript II reverse transcriptase
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Instructions

  1. Processing and preparation of RNA

    • 1

      Harvest cells with an RNA extraction reagent, such as Trizol. 1 ml, is sufficient to collect cells from one well of a six-well tissue culture plate. The cells are thoroughly pipetted up and down to homogenize them and then carry out the extraction procedure as described in the Trizol datasheet. Briefly, extract with chloroform, then centrifuge to separate the phases of DNA, protein and RNA. Recover only the colorless RNA phase and precipitate that with isopropanol. After incubation, centrifuges that and clean up the resulting pellet with several ethanol washes. Then resuspend the pellet in RNase-free water.

    • 2

      For reverse transcription, denature exactly 5 micrograms of RNA at 65 degrees Celsius in a 10 microliter (ul) volume of RNase-free water, then quickly place on ice. For each reaction, combine 10 ul of RNA, 3 ul of 10X PCR reaction buffer, 2.5 ul of 10 millimolar dNTP, 6 l of 25 millimolar magnesium chloride, 1 ul of random primers of 1.8 milligram per milliliter concentration, and 0.5 ul of SuperScript II reverse transcriptase enzyme. Top up each reaction with 17 ul of RNase-free water. Incubate the tubes at room temperature for ten minutes and then at 42 degrees Celsius for an hour to produce the cDNA, then denature at 95 degrees Celsius and quickly ice to stop the reaction.

    • 3

      For a polymerase chain reaction, again set up a reaction mixture in 0.5 ml tubes by combining 6 ul of cDNA made in the reverse transcription reaction, 1.5 ul of 10X PCR reaction buffer, 0.2 microliter of Taq polymerase, 0.5 microliter of reverse primer and also of forward primer, then top up each tube with 10.3 ul of RNase-free water. To run the reactions, set up a thermal cycler (i.e. a machine which performs polymerase chain reactions) for 30 cycles as follows: 95 degrees Celsius for 0.5 minute to denature, followed by 60 degrees Celsius for 45 seconds to anneal, and finally 72 degrees Celsius for 1 minutes to extend the synthesized transcript.

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