The first crucial step in the present protocol is the grinding of the frozen mycelium. Grinding of the mycelium, previously frozen in liquid N2
, by means of Mikrodismembrator II (MD) reduces it, maintained in a frozen state, to a fine and homogeneous powder in a very short time (45 s) thus preventing all degradative processes. MD is a small apparatus where a small steel ball is moved in a bilateral direction inside a small grinding chamber, successfully used for nucleic acids extraction from various biological samples ranging from tumoral tissues [7
] to Bacillus subtilis
]. To maintain the mycelium in a frozen state during grinding, the plastic chamber must be prechilled with liquid N2
. This precaution allows fast, efficient, and highly reproducible grinding of Streptomyces
mycelium. The RNA is then purified according to [3
]. The use of prewarmed SolD to solubilize the mycelium powder prevents the freezing of this buffer and/or the precipitation of some components following the contact with the frozen sample thus allowing a prompt denaturation of cellular RNases. Our purification protocol does not make use of columns thus preventing loss of low M.W. RNA, and the A260/280
of purified RNA appears to be equivalent to that obtained by column procedures. Moreover, the use of an alkaline medium for transfer of the RNA to a filter [5
] improves, by partial RNA hydrolysis, the transfer of high M.W. transcripts. We optimized this step by a preliminary soaking of the gel in NaOH, which allows the gel pH to quickly turn from 6.5 to at least 9.0. Moreover, the use of a NaOH concentration higher than that suggested by [5
] improves glyoxal removal and hydrolysis of long RNA molecules resulting in their efficient transfer to the filter and stronger hybridization signals.
We applied our protocol to the analysis of RNA complementary to the dnaK
operon of S. coelicolor
. Figure shows the pattern of ethidium bromide stained bands seen on the gel and on the filter after the RNA transfer. It clearly appears that the bands corresponding to the 4S and 5S RNA species are almost completely absent in the samples purified by the column procedure (C). However, the intensity of the bands corresponding to larger species is equivalent in the two sets of samples. Figure shows the bands seen after hybridization of the filter to an antisense riboprobe complementary to the 3' distal region of dnaK
operon. This probe hybridizes with transcripts ranging from 4.3 Kb (corresponding to the entire operon [1
] to about 300 nt [6
]. Their expression level is much more enhanced when the temperature of growth is raised from 30°C to 42°C (heat shock treatment [1
]. Comparing our results with those already published on the RNA population complementary to the dnaK
operon during the heat shock response [1
] it clearly appears that the use of our protocol results not only in a highly enhanced intensity of bands associated with the largest transcripts but also in an inversion of the relative intensity of large and small transcripts. Figure shows that the relative intensity of the 4.3 Kb transcript is almost equivalent in samples purified according to either of the two protocols. However, it clearly appears that the hybridization signal of low M.W. transcripts present in the RNA purified with our procedure is much more intense than that seen in the homologous C sample. The latter, although purified by a protocol defined for total RNA extraction (claimed cut-off at 200 nt), results in loss of medium-low M.W. transcripts.
Figure 1 Streptomyces RNA in agarose gel and onto nylon membrane. Glyoxylated RNA (10 μg; purified by means of the Mikrodismembrator (MD) or the columns (C) protocol were fractionated on an agarose gel and transferred to a nylon membrane as described in (more ...)
All this stresses the quality of our protocol for studying dynamic events of RNA metabolism where the relative concentration of high and low M.W. transcripts must be compared.