Isolation of total RNA from Dictyostelium discoideum using Qiagen RNA Isolation Kit
Background:
In the cell transcription produces RNA using DNA as a template. The three main types of RNA that exist in the cell are ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA). mRNA represents the protein coding information from the genes in the DNA. Ribosomal RNA and tRNA are used in the process of translation of the mRNA on ribosomes. In this experiment you will be isolating total RNA from Dictyostelium discoideum using an anion exchange column from Qiagen. You will then use this RNA to amplify a fragment of the cAMP-dependent protein kinase (PKA) gene from D. discoideum by reverse transcriptase-polymerase chain reaction (RT-PCR). Some of you will be using the RNA to amplify the Mms21 gene from D. discoideum.
Laboratory Protocol:
Things to do before starting
If purifying RNA from cell lines rich in RNases, we recommend adding ?-mercaptoethanol (β-ME) to Buffer RLT before use. Add 10 μl β-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing. Buffer RLT containing β-ME can be stored at room temperature (15-25°C) for up to 1 month.
Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96-100%) as indicated on the bottle to obtain a working solution.(Yourprofessor has already done this).
1. Harvest cells according to step 1a:
1a. Cells grown in suspension (do not use more than 1-2 x 107 cells):
Determine the number of cells. Pellet the appropriate number of cells by centrifuging for 5 min at 300 x g in a centrifuge tube (not supplied). Carefully remove all supernatant by aspiration, and proceed to step 2. (Your professor will do this).
2. Disrupt the cells by adding 600 ?l of Buffer RLT.
For pelleted cells, homogenize the cell pellet thoroughly by vortexing vigorously for 3 min.
3. Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge.
Note: When purifying RNA from certain cell lines, precipitates may be visible after addition of ethanol. This does not affect the procedure.
4. Transfer up to 700 μl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at 13,200 rpm. Discard the flow-through.* Repeat as necessary.
If the sample volume exceeds 700 μl, centrifuge successive aliquots in the same
RNeasy spin column. Discard the flow-through after each centrifugation.*
5. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 13,200 rpm to wash the spin column membrane. Discard the flow-through.*
Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure toempty the collection tube completely.
6. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 13,200 rpm towash the spin columnmembrane. Discard the flow-through.
7. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2min at 13,200 rpm towash the spin columnmembrane.
The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere withdownstream reactions.
Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
8. Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min.
Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 8.
9. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 50 μl RNase-free water directly to the spin column membrane. Close the lidgently, and centrifuge for 1 min at 13,200 rpm to elute the RNA.
10. Measure the concentration of your RNA by diluting 1:100 in TE pH 8.0. wavelength would you use is 260.
Questions to be answered:
1. You have isolated total RNA using the Qiagen technique. What does this mean?
2. What would be the major type of RNA present?
3. If you wanted to isolate only mRNA from your lysate, what feature of mRNA would you use to do this?