Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

Use enough TRI Reagent for the sample homogenization. Too small volume of TRI Reagent may result in DNA contamination.

If samples used for the isolation contain organic solvents (ethanol, DMSO), strong buffers, or alkaline solution, DNA contamination may occur.

Incomplete homogenization or lysis of samples may result in low yield of RNA.

The tissues need to be processed or frozen in liquid N2 immediately after removing from the animal to prevent RNA from degradation. If not used immediately, the samples need to be stored at −70°C.

We usually put the tube which contains tissues and TRI Reagent on ice for 2–5 min after homogenizing for 10 s each time. Repeat homogenization for several times or until no tissues are visible.

The homogenizer we used is from Glas-Col, Terre Haute, IN.

TRI Reagent is not compatible with plastic culture plates.

Trypsin digestion of cells may result in RNA degradation.

After addition of the TRI Reagent, we usually put the dish or plate on a shaker for 5–10 min to let cells lyse totally.

Some yeast and bacterial cells may require a homogenizer.

After the cells have been homogenized or lysed in TRI Reagent, samples can be stored at −70°C for up to 1 month.

The chloroform used for phase separation should not contain isoamyl alcohol or other additives.

The mixture also can be put in −4 or −20°C for 1 h.

If the RNA pellets float, after vortexing, centrifuge at 12,000 × g for 5 min at 2–8°C.

Samples can be stored in ethanol at 2–8°C for at least 1 week and up to 1 year at −20°C.

A more complete evaporation of ethanol is required when RNA samples are to be used in RT-PCR. This is especially critical for small volume samples, which may contain a relatively high level of ethanol if not adequately dried. However, do not let the RNA pellet dry completely, as this will greatly decrease its solubility. Do not dry the RNA pellet by centrifugation under vacuum (Speed-Vac).

Incompletely dissolving the final RNA pellet may result in low RNA yield.

Final preparation of RNA is free of DNA and proteins. It should have a A260/A280 ratio of ≥ 1.7.

If the A260/A280 ratio is <1.65, it may be because: (a) the amount of sample used for homogenization may have been too small; (b) samples may not have been allowed to stand at room temperature for 5 min after homogenization; (c) there may have been contamination of the aqueous phase with the phenol phase; (d) the final RNA pellet may not have been completely dissolved; or (e) the water used for dilution of RNA may be acidic. Acidic pH can affect the A280 reading and lowers absorbance ratios. Try to dilute the RNA samples in TE buffer and measure again.

Typical yields from tissues (μg RNA/mg tissue): liver and spleen, 6–10 μg; kidney, 3–4 μg; skeletal muscle and brain, 1–1.5 μg; placenta, 1–4 μg.

Typical yields from cultured cells (μg RNA/106 cells): epithelial cells, 8–15 μg; fibroblasts, 5–7 μg.

The rack should be floated on the water surface. If a plastic PCR rack cannot be found, the plastic racks for 200 μl tips or other racks can be used.

Completely thaw and thoroughly vortex the buffer prior to use.

Spin the tubes containing reagents briefly before opening them.

The extension temperature may be optimized between 37 and 42°C.

Water bath can also be used in stand of a thermal cycler.

It is better to add water first and add DNA polymerase last. Put the DNA polymerase back in the −20°C freezer as soon as possible after using.

Completely thaw and thoroughly vortex the buffer prior to use.

Spin the tubes containing reagents briefly before opening them.

Initial denaturation of longer than 2 min at 95°C is unnecessary and may reduce the yield.

Annealing temperature should be optimized for each primer set based on the primer Tm.

The extension time should be at least 1 min/kb target length.

Housekeeping gene such as β-actin, GAPDH, and so on is also run to demonstrate equal loading.

• (a)

  Initial denaturation: at 95°C for 2 min for 1 cycle

• (b)

  • Denaturation: at 95°C for 0.5–1 min

  • Annealing: at 42–65°C for 0.5–1 min

  • Extension: at 72°C for 1 min/kb

  Amplification (25–35 cycles)

• (c)

  Final extension: at 72°C for 5 min for 1 cycle

• (d)

  Soak at 4°C indefinite for 1 cycle

An example profile of PCR parameters is given as follows.

Make 1% (w/v) agarose gel: weigh 1 g of agarose and pour it into a beaker with 100 ml of 1× TAE or 1× TBE. Put the beaker into a microwave oven. Heat 30 s, take it out to mix, then put it back to the microwave oven. Repeat this step several times until all agarose is melted down. Let it stand at room temperature for several minutes to cool it down until your hand can hold the beaker. Then add ethidium bromide (EtBr) into the gel. EtBr is a potent mutagen that must be handled carefully to avoid skin contact and contamination of the lab. The amount of EtBr to add is as follows: of a 0.5 mg/ml stock solution, add 1/1,000 to your gel. For example, if we go back to our 100 ml gel, then you would add 100 μl of EtBr. Mix gently, then pour the gel onto the casting trays. Let the gel harden at room temperature before using.

The percentage of gel you run mainly depends on the size fragment of PCR product. See for reference (3)

Table 1

% Agarose (w/v)Range of resolution Linear dsDNA (kbp)0.70.8–100.90.5–71.20.4–61.50.2–32.00.1–2Open in a separate window

For reactions containing the 5X Green GoTaq® Reaction Buffer, load amplification reaction onto the gel directly after amplification. Do not need to add any more DNA loading buffer.

2× iQ™ SYBR Green Supermix contains 2× reaction buffer with dNTPs, 50 U/ml iTaq DNA polymerase, 6 mM MgCl2, SYBR Green I, 20 nM fluorescein, and stabilizers.

We use 20 μl as a total volume of one reaction to reduce the cost of experiments. It works well.

Directly add 1 μl of cDNA into the mixture in the wells and make sure no liquid clings to the tip when taking the tip out of the well.

Make sure that the edges of the plate are sealed securely. The film on the four corners is very easily detached.

The parameters for traditional PCR can also be used for real-time PCR.

For each gene to be determined, a test using a few samples is needed before performing many samples to make sure that the parameters are suitable and only one peak is observed in the melting curve.

• (a)

  Initial denaturation: at 95°C for 3 min for 1 cycle

• (b)

  • Denaturation: at 95°C for 10 s

  • Annealing: at 42–65°C for 0.5–1 min

  • Extension: at 72°C for 1 min/kb

  Amplification (40 cycles)

• (c)

  Analysis of the melting curve to confirm the single product amplification during the PCR assay.

• (d)

  Cooling the rotor and thermal chamber at 25°C.

Usually the experimental protocol consists of four programs:

If there is more than one peak in the melting curve, you need to either optimize the PCR parameters or redesign the primers.

The expression level of each gene can also be calculated as copy number or others such as nanomoles, nanograms, and so on. However, a standard curve is necessary for either method.

It is better to have 4–5 points in a standard curve if possible.