RNA handling

Cloning of human RYR1 started with extraction of total RNA. Complementary DNA (cDNA) was synthesised by reverse transcriptase (RT) using extracted mRNA. Synthesised cDNA was used as a template for polymerase chain reaction (PCR). Total RNA was extracted from blood or skeletal muscle samples that were obtained from MHN individuals with informed consent. Blood and muscle samples were stored at -20°C or -80°C, respectively.

2.2.1 RNA extraction from muscle

TRIZOL™ reagent is a mono-phasic solution for the isolation of total RNA from fluids, tissues or cells of a wide range of species including humans. It consists of phenol and guanidine isothiocyanate and is an improved method of the previous one developed in 1987 [Chomczynski and Sacchi, 1987]. During sample homogenisation or lysis, TRIZOL reagent maintains the integrity of RNA and prevents RNase degradation. Added chloroform separates the solution into an aqueous phase and an organic phase. The aqueous phase retains RNA exclusively, but DNA and proteins transfer to the organic phase. RNA in the aqueous phase is recovered by precipitation with isopropyl alcohol. This technique allows the use of small quantities of tissue (50 mg) and cells (5×106), or large quantities of tissue (>1 g) and cells (>107). Total RNA isolated by this reagent is free of protein and DNA contamination, and can be used for several types of experiments including RT-PCR (reverse transcription-polymerase chain reaction). Expected yields of RNA from human skeletal muscle are 1-1.5 μg per 1 mg tissue (manufacturer’s instructions).

As RNA is easily degraded by RNase, which is quite stable, it is important to guard against contamination by RNase and subsequent RNA degradation. All equipment coming in contact with RNA including tips, tubes and glassware, was treated with DEPC-treated water. Sterile water was treated with 0.01% (v/v) diethylpyrocarbonate (DEPC), and plastic and glass items were soaked in DEPC-treated water overnight, and then autoclaved.

fine powder using a mortar designed for rock crushing. Muscle tissues and steel tools were kept cold using liquid nitrogen. The powdered tissue was transferred to a 15 mL round-bottom centrifuge tube containing 0.75 mL of TRIZOL reagent. The solution was homogenised using an ultra-Turax T25 and allowed to stand 5 minutes at room temperature. Addition of 0.2 mL of chloroform followed by centrifugation separated the solution into an aqueous phase and an organic phase. After removal of the aqueous phase, the RNA pellet was collected by precipitation with 0.5 mL of isopropanol. The pellet was washed with 1 mL of 75% ethanol and completely dried using a speedvac for 5 minutes. The RNA pellet was redissolved in 30 μL of DEPC-treated water and was stored at 4°C although RNA samples were normally all used within a few days. Concentrations of RNA samples were calculated by absorption at 260 and 280 nm using an Ultraspec 3000 UV/Visible Spectrophotometer (Pharmacia Biotech) with 100 µL cuvettes. An absorbance of 1.0 at 260 nm is equivalent to an [RNA] of 40 µg/mL while, an A260/280 ratio of 2.0 is considered to be pure RNA [Sambrook, et al., 1989].

2.2.2 RNA extraction from blood

The RYR1 proteins are expressed in white blood cells as well as in skeletal muscle cells and hence blood samples can also be a source of RYR1 mRNA. The total RNA including RYR1 was isolated from human leukocytes using TRIZOL reagent as reported previously [Kraev, et al., 2003]. One half to 1 mL of a blood sample was mixed with three volumes of ice-cold red blood cell lysis buffer containing 150 mM NH4Cl, 10 mM KH2CO3 and 0.1 mM EDTA for 10 minutes on ice. Reaction mixtures

were centrifuged 10 minutes at 1,500 g and the supernatant was removed. The pellet was suspended in 0.75 mL of TRIZOL reagent and homogenised by vigorous pipetting. RNA was extracted using the same procedures as for muscle samples as described in section 2.2.1.

2.2.3 First-strand cDNA synthesis

Total RNA including RYR1 mRNA extracted from blood or muscle samples of MHN individuals was used as a template for synthesis of first-strand of cDNA using SuperScript™ First-Strand Synthesis System. The first-strand cDNA synthesis was

prime the first-strand synthesis for all reactions. One half to 1.0 µg of total RNA was mixed with 1 µL of random hexamers (50 ng/µL), 1 µL of 10 mM dNTP mix and DEPC-treated water to achieve a 13 µL total volume. The mixture was incubated at 65°C for 5 minutes for initial denaturation, and then placed on ice for 1 minute. Seven µL of reaction mixture prepared as a cocktail was then added to the solution. The reaction mixture contained 4 µL of 5× First-strand buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 1 µL of 0.1 M DTT, 1 µL of RNaseOUT™

Recombinant RNase Inhibitor (40 units/µL) and 1 µL of SuperScript™ III RT (200 units/µL). The solution was then incubated at 25°C for 5 minutes for annealing of random hexamers followed by incubation at 50°C for 1 hour for elongation. The reaction was terminated by incubation at 70°C for 15 minutes. The mixture was placed on ice and 1 µL of RNase H (2 units/µL) was added and incubated at 37°C for 20 minutes for degradation of template total RNA. Solutions were stored -20°C until use, and 1-2 µL of 10-fold dilutions were used as a template for subsequent PCR.