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[1.] Iam/Fragment 037 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:03:37 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

Typus
Verschleierung
Bearbeiter
Graf Isolan
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 37, Zeilen: 1ff (complete)
Quelle: Sheikh 2006
Seite(n): 32, 34-35, Zeilen: 32:1-9; 34:13ff - 35:1-4
3.2.2.c. Primer designing

Primers for different genes were designed using the program “Primer Express” (ABI System) and the gene bank data (http://www.ncbi.nlm.nih.gov). All the primer sets used for real-time PCR are listed in the Table 1.

3.2.3 Isolation of total RNA

3.2.3.a. RNA isolation procedure using silica columns

The isolation of total RNA from cultured rat liver hepatocytes, Kupffer cells, myofibroblasts and laser-microdissected samples of the liver was conducted using the NucleoSpin® RNAII kit (Macherey-Nagel) in accordance to the protocol for cultured animal cells.

3.2.3.b. Isolation of RNA by density-gradient ultracentrifugation

Total RNA was isolated from the liver by means of guanidine isothiocyanate extraction, cesium chloride density-gradient ultracentrifugation and ethanol precipitation according to method of Chirgwin (Chirgwin et al. 1979). This method is a versatile and efficient way to extract intact RNA from most tissues and cultured cells, even if the endogenous level of RNase is high.

Cell lysis: The cells were rapidly lysed in guanidine isothiocyanate-containing buffer, which ensures inactivation of RNases. The lysates were layered onto a CsCl gradient and spun in an ultracentrifuge. Proteins remain in the aqueous guanidine portion, DNA bands in the CsCl, and RNA settle down at the bottom of the tubes as a pallet [sic]. The RNA was recovered by dissolving the pellet. The recovery of RNA was usually excellent if the capacity of the gradient did not exceed.

Homogenization of the tissue sample: About 100 mg of frozen tissue was homogenized with Ultra-Turrax TP 18/10 homogenizer 3 times for 10 sec each in 3 ml of ice-cold GITC buffer with freshly added Antifoam A (Sigma). The homogenates were centrifuged for 10 min at 3,500 rpm in a Rotixa /RP centrifuge (Hettich) at 4°C to pellet connective tissue and large cell debris.

CsCl gradient and ultra centrifugation: To prepare the gradient 2 ml of CsCl buffer was poured into 5-ml polyallomer ultracentrifuge tubes (6 per preparation). The cleared guanidine lysed samples were carefully layered on top of the CsCl buffer.


Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299

[Page 32]

3.2.1.IV Primers designing

Primers for different genes were designed using the program “Primer Express” (ABI System) and the gene bank data (http://www.ncbi.nlm.nih.gov). All the primer sets used for real-time PCR are listed in the Table 1.

3.2.2 Isolation of total RNA

3.2.2.I RNA isolation procedure using silica columns

The isolation of total RNA from cultured rat hepatocytes was conducted using the NucleoSpin® RNAII kit (Macherey-Nagel) in accordance to the protocol for cultured animal cells.

[Page 34]

3.2.2.IV Isolation of RNA by density-gradient ultracentrifugation

Total RNA was isolated from the liver, the skeletal muscle and extrahepatic organs by means of guanidine isothiocyanate extraction, cesium chloride density-gradient ultracentrifugation and ethanol precipitation according to method of Chirgwin (Chirgwin et al, 1979). This method is a versatile and efficient way to extract intact RNA from most tissues and cultured cells, even if the endogenous level of RNase is high.

Cell lysis

The cells were rapidly lysed in guanidine isothiocyanate-containing buffer, which ensures inactivation of RNases. The lysates were layered onto a CsCl gradient and spun in an ultracentrifuge. Proteins remain in the aqueous guanidine portion, DNA bands in the CsCl, and RNA settle down at the bottom of the tubes as a pellet. The RNA was recovered by dissolving the pellet. The recovery of RNA was usually excellent if the capacity of the gradient does not exceed.

Homogenization of the tissue sample

About 100 mg of frozen tissue was homogenized with ultra-turrax TP 18/10 homogenizer 3 times for 10 sec each in 3 ml of ice-cold GITC buffer with freshly added antifoam A (Sigma). The homogenates were centrifuged for 10 min at 3,500 rpm in a Rotixa/RP centrifuge (Hettich) at 4°C to pellet connective tissue and large cell debris.

[Page 35]

CsCl gradient and ultra centrifugation

To prepare the gradient 2 ml of CsCl buffer was poured into 5-ml polyallomer ultracentrifuge tubes (6 per preparation). The cleared guanidine lysed samples were carefully layered on top of the CsCl buffer.


Chirgwin JM, Przybyla AE, MacDonald RJ and Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294- 5299, 1979.

Anmerkungen

Slightly adapted, but otherwise nearly identical, though not marked as a citation.

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(Graf Isolan) Schumann


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