Can you simplify these methods for me please. The methods are in bold. Please su

Question

Can you simplify these methods for me please. The methods are in bold. Please summarize all three methods please.

Cell Lines and Cell Culture. The ER-positiveHBC cell line MCF-7and the ER-negative HBC cell line MDA-MB-23 1 were kindly provided by Dr. Marc Lippman (Lombardi Cancer Center, Washington, DC). ER-negative HBC cell lines SKBR-3 and MDA-MB-435 were obtained from Dr. Steven Byers (Lombardi Cancer Center, Washington, DC) and Dr. Janet Price (Uni versity of Texas M. D. Anderson Cancer Center, Houston, TX), respectively. ER-negative HBC cell line MDA-MB-468 was kindly provided by Dr. Ann Hamburger (University of Maryland Cancer Center, Baltimore, MD). MDA MB-23l cells stably transfected with ER were as described previously (10). Cells were cultured routinely in DMEMIHam's F-12 medium (1:1) supple mented with 5% fetal bovine serum as described previously (5).

Retinoids and Growth Experiments. All-trans-RAwas purchasedfrom Sigma Chemical Co. TFAB, SR1 l003/Am580, and SRI 1262 were as de scribed previously (12). RA is an activator of RARa, RAR@, and RARy. TTAB activates the RARs only, whereas SRi 1003 is a selective activator of RARct. SR11262 is a selective activator of RARfJ/y. For growth experiments,cells were cultured as described above and treated with various retinoids for periods of 3, 6, and 9 days in the same medium. Control cells were treated with vehicle alone, and the medium and the retinoids were changed every 2 days. Cells were trypsinized and counted using a hemocytometer.

Southern and Northern Blot Analysis. Genomic DNA extractionsfrom various HBC cell lines, restriction digestion, and Southern blots were per formed according to standard procedures (1 3). RNA extraction and Northern blot analysis were performed essentially as described previously (14). The cDNA probes for hRARa exons 1 and 2 were as described previously (11). The plasmid containing the cDNA fragment of the human acidic ribosomal phosphoprotein P0 (clone p36B4) was as described previously (15). The cDNAs encoding human homologues of the krox proteins (egrl, egr2, and egr3; Ref. 16) were kindly provided by Dr. Vikas Sukhatme (Harvard Medical School, Boston, MA). The probe labeling was according to the random primer method of Feinberg and Vogelstein (17). The hybridization and washing conditions were as described by Sambrook et a!. (13). Promoter Deletional Constructs. RARa promot

Explanation / Answer

Answer:- These methods seems to be published in a research article. This is a typical research literature. I will try to simplify it as much as possible.

1. Cell line and cell culture:- Here the researchers are describing about the human breast cancer cell lines they have used for this study. They have used Estrogen receptor (ER) positive and as well as negative cell lines. MCF-7 is a ER +ve cell line. The name MCF-7 comes from the institution from where it was isolated from a cancer patient. MCF stands for Michigan cancer foundation. Likewise MDA MB-231 comes from M.D. Anderson Cancer center. MDA-MB 231 being ER -ve is more aggressive in nature and the researcher must have used it for comparison purposes.

They have also used 3 other ER-ve cell lines named as SKBR3, MDA MB 435 and MDA MB 468. The reason for using them may be the differential aggressive nature of all of these cell lines.

Of all the cell lines only MDA MB-231 was cloned for stable expression of ER in the cells. This means that they have artificially introduced ER protein in the cells and made the cells stable for its its expression. Therefore this cell line which was ER-ve earlier will now be ER+ve because of this genetic manipulation.

In the last line researcher has described that how he has grown the cells and in which medium. These cells were grown in a higly enriched medium which is a mixture of DMEM and Hams F12. DMEM stands from dulbecoos modified eagle medium and it was mixed in 1:1 ratio with Hams F12. This medium contains the nutrient required for the growth of the cells in in vitro. i.e. laboratory conditions. This medium needs to be supplied with serum for prooer growth and here they have used 5% FBS. FBS stands for fetal bovine serum.

Retinoids and Growth Experiments:-

Here they have used all trans retinoids for cell growth experiments. They have used these trans retinoids because it activates certain receptor proteins such as RARa, RAR@, RARy and others. The retinoids they used are RA, TTAB, SRi1003 and SR11262. In growth assay they cultured the cells as described in experimentation no. 1. Then they treated the cells with these retinoids to see their effect on the cell growth. The experiment was carried out for the period of 3, 6 and 9 days in the same medium. They used a control where they didn't treat the cells with retinoid, so it was a negative control for comparison purposes. After the desired time was over they used trypsin which an enzyme that cleaves the arginine and lysine residue of the cell attached to the plate surface so that it is dissociated from the plate and used it for counting the cells. Counting was done to measure the effect of retinoids on cell growth.

Southern and Northern Blot Analysis:-

In this experiment researcher has done southern blotting that is done by isolating DNA from the breast cancer cells and also the northern blotting by isolating mRNA from the human breast cancer cells. They have used some cDNA probes. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. They have also used a plasmid containing the cDNA fragment of the human acidic ribosomal phosphoprotein P0. plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. It has a property of replicating seperately and is usd to intoduce genes in the host organisms which is usually a bacteria. All the other techniques and details in this experiment is almost impossible to describe as they have give reference to the previous papers which are impossible to track from here. Also the method mentioned in this question is not complete and ends abruptly.

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