A rapid method of analysis for determining the identity of neural stem cells (NSCs) and their sublineages involves the early detection of differentiation markers tracked at the RNA level. This protocol follows methodologies described in the PureLink™ RNA Mini Kit manual for isolating total RNA from neural stem cells (NSCs), followed by cDNA synthesis using Superscript® III reverse transcriptase. The following protocol gives you a step-by-step procedure for template preparation required for RT-PCR or qPCR.
- Neural stem cells
- PureLink™ RNA Mini Kit (Cat. no. 12183-018A)
- RNA Lysis Solution
- Wash Buffer I
- Wash Buffer II
- RNase-free water
- RNA spin cartridges
- Collection tubes
- RNA recovery tubes
- Superscript® III First Strand Synthesis SuperMix (Cat. no. 18080-400)
- Superscript® III/RNaseOUT™ Enzyme Mix
- 2X First-strand Reaction Mix
- Annealing Buffer
- 50 mM Oligo(dT)20
- Random hexamers (50 ng/mL)
- β-Mercaptoethanol (Cat. no. 21985-023)
- TrypLE™ Express Stable Trypsin Replacement Enzyme (Cat. no. 12604-013)
- Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca2+ and Mg2+ (Cat. no. 14190)
- Ribonuclease H (RNase H) (Cat. no. 18021-071)
- 10X BlueJuice™ Gel Loading Buffer (Cat. no. 10816-015)
- Table-top centrifuge
Important: Perform all steps on ice unless noted otherwise. For all incubations, heat the thermocyclers in advance. Pre-chill all reagents and thaw all frozen reagents and cells immediately prior to use. Use RNase-free pipette tips with aerosol barriers.
- Prepare RNA Lysis Solution by adding 10 μL β-mercaptoethanol per mL of RNA Lysis Solution.
- Remove media from T-25 flasks, rinse once with Dulbecco’s phosphate-buffered saline (D-PBS) and treat cells with 1 mL of pre-warmed TrypLE™ reagent for 10 minutes at 37°C.
- Harvest the cells and place them into 15-mL centrifuge tubes. Take 100 μL of the sample and obtain a viable cell count.
- Centrifuge the cells in a tabletop centrifuge for 7 minutes at 100 × g. Discard the supernatant.
- Freeze the cells overnight in a -70°C freezer.
- Allow the cell pellet to thaw. Add 0.5 mL of RNA Lysis Solution for each T-25 flask harvested for the pellet (0.5 mL per 2 × 106–5 × 106 cells). Pipet the cells ~20 times until the pellet is disrupted.
- Transfer 0.5 mL of cell lysis solution to 1.5-mL RNase-free microcentrifuge tubes and centrifuge at room temperature for 2 minutes at 12,000 × g (12,000 rpm).
- Add 0.5 mL of 70% ethanol to each tube, and vortex the suspension 5–10 times.
- Apply a 600 μL aliquot of sample to the RNA Spin Cartridge. Centrifuge at room temperature for 15–30 seconds at 12,000 × g, then discard the flow-through. Continue applying 600 μL aliquots of the same RNA sample to the spin cartridge until the entire sample has been processed.
- Add 700 μL Wash Buffer I to the spin cartridge and centrifuge at room temperature for 15–30 seconds at 12,000 × g. Discard the flow-through and the tube. Place the spin cartridge into a clean 2 mL RNA Wash Tube.
- Add 500 μL Wash Buffer II (containing ethanol) to the spin cartridge and centrifuge at room temperature for 15–30 seconds at 12,000 × g. Discard the flow-through. Centrifuge for 1 minute to dry the cartridge.
- Place the cartridge into an RNA Recovery Tube. Add 40 μL of RNase-free water to the cartridge, and let it stand for 1 minute. Centrifuge the cartridge at room temperature for 2 minutes at 12,000 × g. Add an additional 40 μL of RNase-free water to the cartridge and repeat the step. Yield should be about 60–300 μg total RNA.
Note: Always allow time for the RNase-free water to percolate into the cartridge bed. Do not spin the cartridge immediately because it may result in partial recovery and alter the yield of RNA. To recover more RNA, add an additional 40 μL of RNase-free water to the cartridge and repeat the last step for a third time.
Determining RNA Quality
- Measure ratio of absorbance at 260 nm and 280 nm by analyzing 1 μL of the RNA sample using a NanoDrop™ spectrophotometer. Conduct readings three times, and use the average as the final value. Wipe down the analysis stage with a lab tissue wetted with DEPC water before and after measuring each RNA sample. The A260/280 of pure RNA is ~2. Note: The yield and quality of the isolated RNA depends on the type and age of the starting material, in addition to how the material was collected and preserved.
- Prepare the RNA samples for RNA gel analysis as follows:
Component Amount RNA sample 1 μL 2X BlueJuice™ gel loading buffer 1 μL DEPC-treated water 8 μL
- Mix the components and load the samples onto individual wells of an agarose gel. Use 10 μL of 0.1 kb and 1 kb molecular weight markers to estimate the molecular weight size of ribosomal RNA bands. Use 10 μL DEPC water for empty wells. Run samples for 30 minutes, visualize the bands on an UV light box, capture the gel image, and perform band intensity measurements.
Store RNA samples at -70°C or process it further for cDNA synthesis.
First-Strand cDNA Synthesis
This protocol follows the methodologies described in the instructions for Superscript® III First Strand Synthesis SuperMix.
- Mix and briefly centrifuge each component before use. Pre-heat the thermocycler to 65°C.
- Combine the following components on ice in a 0.2-mL thin-walled PCR tube. Use a volume containing up to 1 μg of total RNA for the reaction.
Component Amount Annealing buffer 1 μL Random hexamer(50 ng/μL) 1 μL RNA (1 μg) x μL DEPC-treated water to 8 μL
- Incubate the reaction in the thermocycler at 65°C for 5 minutes, and then immediately place on ice for at least 1 minute. Collect the contents of the tube by brief centrifugation.
- While the tube is on ice, add the following components to the tube:
Component Amount 2X First–Strand Reaction Mix 10 μL SuperScript® III/RNaseOUT™ Enzyme mix 2 μL
- Vortex the sample briefly, and collect the contents by brief centrifugation.
- Incubate the tube at 25°C for 10 minutes.
- Incubate the tube at 42°C for 50 minutes.
- Terminate reaction by incubating at 85°C for 5 minutes, then chill the tube on ice.
- Add 1 μL of RNAse H to the sample, and incubate at 37°C for 20 minutes.
- Store the cDNA samples at –20°C.