RNase solution preparation

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

A Working Solution of the stain should be prepared just before use, by combining 10 ml of 0.1% Triton X-100 in phosphate-buffered saline, 2 mg RNase (DNase-free, Sigma), and 200 pi PI solution (Sigma, 1 mg/ml in distilled water). Stock solutions of Triton X-100 and PI should be stored at 4°C. Centrifuge the ethanol-fixed cells (200 x g for 5 min) and discard the supernatant, removing it completely. 

Remove excess PBS and immediately apply 50 pi double-FAM LNA (see Note 2) probe solution and gently shield with cover glass (see Note 3). The probe solution is prepared as follows denature LNA probe and dilute the probe in Exiqon ISH buffer. For example, for 2 ml hybridization mix containing 20 nM double-FAM-labeled miR-21 LNA probe (from 25 pM probe stock), transfer 4 pi into the bottom of a 2 ml nonstick RNase-free tube and place the tube at 90°C for 4 min. Spin down shortly using a tabletop centrifuge, and immediately... 

This multiprobe RPA offers the advantages of its sensitivity and capacity to simultaneously quantitate several mRNA species in a single sample of total RNA. This allows comparative analysis of different mRNA species within samples, moreover, by incorporating probes for housekeeping gene transcripts, the levels of individual mRNA species can be compared between samples. Furthermore, the assay is highly specific and quantitative owing to the RNase sensitivity of mismatched base pairs and the use of solution-phase hybridization driven toward completion by excess probe. Finally, the multiprobe RPA can be performed on total RNA preparations derived by standard methods from either frozen tissues or cultured cells. 

Ribonuclease A (RNase A) is dissolved at 1 mg/ml in water, and stored at -20°C. To inactivate the possible contaminated DNase completely, RNase A solution is sometimes boiled once when prepared. 

DEPC-treated water should be used for all RNA preparation solutions, gloves should be worn at all times, and RNase-free tips and tubes used. It is not necessary to purify mRNA, since total RNA prepared from freshly isolated or even frozen (liquid nitrogen or-80°C) lymphocytes is pure enough to carry out the RT-PCR reactions. A number of commercial kits are now available for RNA isolation (e.g., Stratagene), and use of one of these is recommended. Alternatively the single-step guanidimum thiocyanate/phenol extraction method of Chomczynski and Sacchi (23), on which most kit protocols are based, should be employed. If the lymphocyte numbers are low (<5 x 106), carrier RNA (100 pg of 16S ribosomal RNA) can be added at the start of this procedure to aid recovery. The preparation ends with a precipitation step using isopropanol, and the RNA may stored at -20°C in this form until required. 

Another issue that should be considered carefully is the purity of nucleic acids used at each step. Since it is known that RNA can self-cleave and ligate, DNA produced from PCR that is entering the next cycle of selection should be size-purified by native PAGE (Section 8.3.1.5). Finally, all solutions should be prepared from DNase/RNase-free reagents in DEPC H20, 0.2 pm filtered, and stored at 4 °C (buffers) or —20 °C (especially nucleotide solutions). 

To resolve folded and unfolded conformations of the 387 nt Tetrahymena ribozyme, we use 8% acrylamide (29 1 mono bisacrylamide) in 34 mMTris, 66 mM Hepes (pH 7.5), 0.1 mM EDTA, and 3 mM MgCl2 (THEM3). Hepes is used instead of borate to maintain the native structure of the ribozyme (Buchmueller and Weeks, 2004 Pyle et ah, 1990), whereas the MgCl2 concentration is chosen to be just sufficient to maintain the RNA in its folded state. Twenty-five milliliters of acrylamide solution per gel is prepared and degassed using RNase-free water. 200 /iL 10% ammonium... 

Residual RNA in a DNA preparation can be removed by treatment with ribonuclease (RNase). RNase A, which is free of DNase, is available commercially, or the contaminant DNase in the crude RNase A solution can be heat inactivated by heating RNase A solution (10 mg/mL in 10 mM Tris-Cl, pH 7.5, 15 mMNaCl) at 100°C for 15 minutes [4], DNA solution in TE at a concentration of at least 100 pg/mL is treated with RNase to a final concentration of 1 pg/mL followed by incubation at 37°C for 1 hour [3], RNase... 

For first-strand cDNA synthesis SUPERSCRIPT II RT reverse transcriptase (200 U/pL) supplemented with 5X RT buffer (250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15mM MgCl2) (Gibco-BRL), random hexamers (50 ng/pL), 0.1 M DTT, 10 mM dNTP mix, and DEPC-treated water. These solutions should be prepared as RNase-free. We utilize solutions supplemented with cDNA synthesis kit (Gibco-BRL)... 

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

The extreme sensitivity of RNA to the ubiquitous inter- and intracellular nudeases (for example on the skin of the investigator) makes special precautions necessary for effective RNA preparations. The use of disposable containers is recommended, or glassware that has been soaked in dilute hydrochloric acid and rinsed with autoclaved distilled water. Disposable gloves must be worn in all procedures where RNA is handled, or is likely to come into contact with RNA, such as solutions, chemicals, glassware, spatulas etc. Buffers for RNA work should be prepared from reagents reserved for this purpose, and stored separately. Buffers can be treated with 0.2 % (v/v) diethylpyrocarbonate (care - this is carcinogenic) and autodaved to inactivate RNases, or at least those with adive site histidines. Since most nucleases require Mg2 for activity, the addition of EDTA in mM concentrations to solutions is also recommended. 

All solutions used for reverse transcription must be free of RNase. If all solutions are prepared with RNase-free water in disposable plastics and no contact with RNase-contaminated spatula or pH probes has occurred, the solutions need not be treated any further. Otherwise, to decontaminate the solutions add diethyl pyrocarbonate (DEPC) (e.g., Sigma, St. Louis, MO) to the solution to a final concentration of 0.1%, shake, let sit overnight with loosened caps, and then autoclave for 15 min. Note that DEPC might be carcinogenic and that solutions containing Tris cannot be decontaminated with DEPC, but must be prepared with special caution to prevent any RNase contamination. 

Solutions (including doubled distilled water, buffer and culture medium) should be sterile and DNase- and RNase-free grade and stored at 2-8°C (+4°C) after opened. Unless stated otherwise, all solutions should be prepared in water that has a resistivity of 18.2 MU cm and total organic content of less than five parts per billion. This standard is referred to as doubled distilled water in this text. 

Successful hybridizations depend critically on the quality and purity of both the starting RNA and of the labeled end-product. As with all RNA work, RNase-free reagents should be used throughout. Purified ddH20 used to prepare solutions should be treated with 0.1% diethylpyrocarbonoate (DEPC) overnight and then autoclaved. 

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