After a prehybridization step in Hybridization Cocktail 50% Formamide (AMRESCO, Solon, OH) for 1 h at 58 [degrees]C, colonies and hemocytes were incubated with sense and antisense probes (2 [micro]g/ml biotin-labeled riboprobe
in Hybridization Cocktail) overnight at 58 [degrees]C.
Polyvalent detection by using the non-radioactive molecular hybridization technique has been performed in two different forms: first, by a mixture of the specific single probes in the hybridization solution, and secondly, by using a unique riboprobe
, called polyprobe, that contains partial nucleic acid sequences of different viruses cloned in tandem (PEIRO et al.
The cRNA product was reamplified, cleaned (QIAquick PCR Purification Kit; Qiagen, Valencia, CA), and transcribed using the Riboprobe
In Vitro Transcription System (Promega Corp.
Because of the nested set structure of the coronavirus genomic RNA (genRNA) and messenger RNAs (mRNAs), the nucleocapsid riboprobe
would detect all viral RNAs (18).
To investigate OMT downregulation of the endogenous OMT mRNA steady state levels, northern blot analysis was performed with an omt antisense riboprobe
with the third leaf of those five plants that maintained reduced OMT activity at the 15-leaf stage.
To ensure the integrity of template RNA in each tissue section, a control riboprobe
targeting ubiquitous cellular U6 RNA was applied in parallel reactions.
elegans, with a riboprobe
concentration of 1 ng [[micro]l.
For detection of WNV nucleic acid, an antisense digoxigenin- labeled riboprobe
complementary to nt 4966-5439 of WNV strain NY1999 was generated from plasmid pWNNY-88B-14 (W.
5 mg/mL yeast RNA) containing each riboprobe
at 10,000 cpm/[micro]L, and hybridization was performed at 55[degrees]C overnight.
The deleted vector was transcribed by the Riboprobe
in vitro Transcription System (Promega).
To determine if the abnormalities in neural crest-derived melanocytes and increased apoptosis in neural regions could be attributed, at least in part, to changes induced in the expression of the neural crest-specific transcription factor Xslug, nonisotopic in situ hybridizations with a digoxigenin-labeled riboprobe
directed against Xslug cDNA (Mayor et al.
In situ hybridization with an usherin-specific riboprobe
(nN2A) confirms that usherins are expressed both in planulae and in juvenile polyps (Fig.