total rna isolation from cockroach - (Nov/14/2006 )
hi... can somebody help me with the exact protocol for isolating total RNA from cockroach...
-centaur-
QUOTE (centaur @ Nov 14 2006, 07:16 PM)
hi... can somebody help me with the exact protocol for isolating total RNA from cockroach...
Hi cenntaur,
The "exact protocol" would be that applying to your needs.
Here you are a link that may orient you for the beginning
http://www.entomology.cornell.edu/Faculty_...f/Scott/121.pdf
RNA isolation chapter says
"Total RNA was extracted using standard methods (Sambrook et al., 1989) with some modifications. Briefly, the abdomen of a male German cockroach was amputated, homogenized on ice in 0.4 ml of cold guanidine thiocyanate buffer (4 M, pH 7.0, containing 25 mM sodium citrate, 0.5% N-lauroyl-sarcosine and 0.1 M beta-mercaptoethanol), then incubated with 0.5 ml phenol-chloroform (1:1) at 65°C for 15 min with occasional inversion. The mixture was put on ice for 10 min
and then centrifuged for 10 min. The supernatant was removed, mixed with 0.4 ml of 4.0 M lithium
chloride, chilled at –70°C for 30 min and centrifuged for 20 min (15,000g) at 4°C. The pellet was
dissolved in 0.2 ml of TENS buffer (0.1 M Tris buffer, pH 7.4, 50 mM sodium chloride, 0.1 M EDTA, 0.2% SDS) containing 2 ml proteinase K (>400 U/ml) (Gibco/BRL), incubated at 37°C for 0.5–1.5 h and extracted twice with an equal volume of phenol-chloroform. RNA was precipitated with 0.1 volume of 3 M sodium acetate and 2 volumes of absolute ethanol. The pellet was dissolved
in water and stored at –70°C for further use.
In another cockroach study one uses RNAqueous-4PCR RNA Isolation Kit Ambion, or Aurum total RNA fatty and fibrous tissue kit Bio-Rad. You may try one of these kits.
More important for maximizing the yield/quality of your RNA is to find the most appropriate method of tissue disruption for your specific starting material. This can be problematic when tissues are hard. Commonly, freeze the tissue in liquid nitrogen or on dry ice, then ground your samples with a mortar and pestle into a fine powder, then proceed with denaturation. It's time consuming and laborious procedure.
RNAlater from Ambion is a tissue storage reagent that stabilizes RNA in unfrozen samples, allowing you to process them later. Harvest tissue pieces and put them in RNAlater for storage (frozen or unfrozen). It is compatible with several isolation reagents such as TRI, Trizol, etc.
Hope it helps
-lillymay-
QUOTE (lillymay @ Nov 15 2006, 06:00 AM)
QUOTE (centaur @ Nov 14 2006, 07:16 PM)
hi... can somebody help me with the exact protocol for isolating total RNA from cockroach...
Hi cenntaur,
The "exact protocol" would be that applying to your needs.
Here you are a link that may orient you for the beginning
http://www.entomology.cornell.edu/Faculty_...f/Scott/121.pdf
RNA isolation chapter says
"Total RNA was extracted using standard methods (Sambrook et al., 1989) with some modifications. Briefly, the abdomen of a male German cockroach was amputated, homogenized on ice in 0.4 ml of cold guanidine thiocyanate buffer (4 M, pH 7.0, containing 25 mM sodium citrate, 0.5% N-lauroyl-sarcosine and 0.1 M beta-mercaptoethanol), then incubated with 0.5 ml phenol-chloroform (1:1) at 65°C for 15 min with occasional inversion. The mixture was put on ice for 10 min
and then centrifuged for 10 min. The supernatant was removed, mixed with 0.4 ml of 4.0 M lithium
chloride, chilled at –70°C for 30 min and centrifuged for 20 min (15,000g) at 4°C. The pellet was
dissolved in 0.2 ml of TENS buffer (0.1 M Tris buffer, pH 7.4, 50 mM sodium chloride, 0.1 M EDTA, 0.2% SDS) containing 2 ml proteinase K (>400 U/ml) (Gibco/BRL), incubated at 37°C for 0.5–1.5 h and extracted twice with an equal volume of phenol-chloroform. RNA was precipitated with 0.1 volume of 3 M sodium acetate and 2 volumes of absolute ethanol. The pellet was dissolved
in water and stored at –70°C for further use.
In another cockroach study one uses RNAqueous-4PCR RNA Isolation Kit Ambion, or Aurum total RNA fatty and fibrous tissue kit Bio-Rad. You may try one of these kits.
More important for maximizing the yield/quality of your RNA is to find the most appropriate method of tissue disruption for your specific starting material. This can be problematic when tissues are hard. Commonly, freeze the tissue in liquid nitrogen or on dry ice, then ground your samples with a mortar and pestle into a fine powder, then proceed with denaturation. It's time consuming and laborious procedure.
RNAlater from Ambion is a tissue storage reagent that stabilizes RNA in unfrozen samples, allowing you to process them later. Harvest tissue pieces and put them in RNAlater for storage (frozen or unfrozen). It is compatible with several isolation reagents such as TRI, Trizol, etc.
Hope it helps
This is the first time am gonna work with cockroach
lil apprehensive bout it...my lab uses the eppendorf kit...since i am interested in the tropomyosin gene i was wondering if i can homogenize the entire roach....
and hey...thanks a million ton for the tip
-centaur-