RNA extraction protocols

A wide variety of reagents are available for RNA extraction. One of the most commonly used methods is based upon that of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987, Chomczynski, 1993), using a monophasic lysis reagent containing phenol, guanidinium salts and solubulizing agents (available under different trade-names from a variety of suppliers). This approach enables simultaneous extraction of RNA, DNA and protein, and is effective for even small tissue samples.

Use of such monophasic lysis reagents is particularly effective on ocular tissues, including retina, RPE, lens as well as whole eye, and has also been applied to a diverse range of tissues throughout the body. When using a different tissue type for the first time, it is always worth conducting a test extraction, evaluating RNA quantity and quality to ensure that the RNA extracted suitable for subsequent studies. Using a monophasic lysis reagent, it is possible to routinely extract 10-15 pg of high quality total RNA from paired whole eyes, and 1-5 pg of total RNA from paired retinae. When studying models of retinal degeneration, these yields decrease, with 0.5-2 pg total RNA being more typical.

Sham 30 min 75 min

Light treatment

Sham 30 min 75 min

Light treatment

Figure 6.5

C-fos induction in wildtype retina, dissected from tissue preserved in RNAlaterâ„¢ (Ambion). Peak expression of c-fos was found 30 min after light pulse administration. Significant differences were assessed using one-way ANOVA, F211 = 13.56, P<0.01 (n = 4-5 per group).

Analysis of RNA samples using a microfluidic system, such as the Agilent bioanalyzer, enables RNA quality to be assessed with minimum loss of valuable RNA (Figure 6.6). Such lab-on-a-chip methods typically utilize just 500 ng of RNA, and enable a quantitative measure of RNA quality as well as quantity.

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