Tissue Collection

1. Sterilize and clean with an RNase decontamination solution any tools used for the dissection of the experimental tissue (see Note 3) to ensure that no exogenous RNases are introduced.

2. To preserve RNA integrity, snap-freeze the tissue very rapidly after dissection. Alternatively, store the tissue in an RNA-preservation medium such as Ambion's RNAlater. (For further information, see RNAlater protocol available from Ambion website: www.ambion.com/techlib/prot/bp_7020.pdf.) The latter is a high-salt buffer that precipitates proteins, and as long as tissue samples are small enough to enable penetration, this offers a simple method of preventing any further enzymatic reactions from occurring.

Fig. 1. Flowchart of RNA extraction, from tissue to purified total RNA.

3.1.1. Samples From Environment-Sensitive Tissues

RNAlater is of particular use with scenarios where gene expression may change rapidly, providing a stable medium for later tissue dissection (see www.ambion.com/techlib/prot/bp_7020.pdf.).

1. Place the dissected tissue in RNAlater in darkness.

2. Store in a light-tight container at 4°C if dissection is to be carried out a few days later. For prolonged storage keep the sample at -20 or - 80°C.

In circadian biology, assaying changes in gene expression following light pulsing provides an obvious example. Figure 2 shows qPCR data from c-fos induction in the mouse retina collected in this manner (data courtesy of D. Elfant, unpublished observations). Despite storage at 4°C prior to dissection, the characteristic rapid light induction associated with this immediate-early gene is unaffected by dissection in the light (4).

3.1.2. Hypothalamic Tissue Punches

Extraction of small samples such as hypothalamic tissue punches are also feasible using a phenol-chloroform approach, although pooling of samples may be necessary to obtain enough RNA.

1. Rapidly remove the brain from anesthetized animals and snap-freeze in isopentane at -60°C on dried ice for 20 s.

2. Cut a 1-mm coronal section at the level of the optic chiasm.

Sham 30 min 75 min

Light Treatment

Fig. 2. Collection of light-sensitive tissues, such as retinae, may be facilitated by an RNA stabilization medium, such as RNAlater (Ambion). This enables tissue to be collected in darkness, and dissected at a later date in light, without the problems associated with freeze-thawing. This figure shows c-fos induction in the murine retina, where whole eyes were collected in darkness with an infrared viewer, and dissected out under light 48 h later.

Sham 30 min 75 min

Light Treatment

Fig. 2. Collection of light-sensitive tissues, such as retinae, may be facilitated by an RNA stabilization medium, such as RNAlater (Ambion). This enables tissue to be collected in darkness, and dissected at a later date in light, without the problems associated with freeze-thawing. This figure shows c-fos induction in the murine retina, where whole eyes were collected in darkness with an infrared viewer, and dissected out under light 48 h later.

3. Take a suprachiasmatic nuclei (SCN) punch using a flat-tipped 25-G needle (diameter approx 1 mm) and store the tissue on dried ice until phenol-chloroform extraction.

Figure 3 shows the SCN of the hypothalamus. Figure 3A shows staining of the SCN for mPer2 mRNA as assayed by in situ hybridization (images courtesy of Marta Muñoz), illustrating the region of the SCN sampled. Figure 3B shows mPer2 expression data from six pooled SCN assayed by quantitative real-time PCR using SYBR Green I, as described previously (5). Although this represents quite a gross approach to tissue sampling in comparison with more refined techniques such as laser capture microdissection, it illustrates that even small tissue samples may provide enough RNA for reliable quantitative analysis.

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