FORMAzol as an RNA storage medium: a cautionary note when performing RT-PCR.

The labile nature of RNA demands careful procedures for its extraction, purification, and storage. Generally, RNA is solubilized in aqueous buffers or organic solvents, or precipitated with alcohol and then kept at -20 degrees C or colder. A commercially available product for RNA storage is FORMAzol(R) (Molecular Research Center). We began using FORMAzol because the Application Notes from the Product Description sheet claims that reverse-transcription (RT) is not inhibited so long as FORMAzol does not exceed 5% (v/v) in the reaction mix. This is ostensibly more convenient than having to precipitate RNA, resolubilizing it in water or buffer, and then proceeding with RT-PCR. However, amplicon yields for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were poor when using RNA directly from FORMAzol, even though its final concentration was typically much less than 5%. By contrast, satisfactory RT-PCR products were obtained with RNA stored frozen in water or that had been resolubilized from alcohol precipitates. When RT-PCR was then performed on ethanol-precipitated and resolubilized RNA from FORMAzol, yields of GAPDH amplicons were acceptable. Although a revision to the FORMAzol Product Description sheet is now available at the manufacturer's website (http://www.mrcgene.com/formazol.htm), if users of the product implicitly follow the directions found in the package insert sheet-not being aware of the inhibitory effects of formamide (the denaturant in FORMAzol)-unsatisfactory results may be obtained from RT-PCR experiments. It is suggested that FORMAzol only be used for RNA storage and that RNA be precipitated with alcohol, washed, and resolubilized prior to use.

Pancreatic regeneration (reg) gene expression in a rat model of islet hyperplasia.

BACKGROUND: After pancreatectomy, regeneration of acinar and islet elements occurs. Recent data from a model of islet hyperplasia in the hamster suggested that induction of a local pancreatic factor stimulates islet formation. We postulate that the reg gene may be this factor. METHODS: We studied reg expression during induction of islet growth by using a similar model in the rat. Rats underwent surgical wrapping of the splenic lobe of the pancreas or sham operation. RESULTS: At 2 days ductular proliferation and immunohistochemical evidence of insulin within ductular epithelia were evident in the wrapped lobe. By 14 and 56 days islet number per square millimeter increased 63% and 43%, respectively. Reg mRNA levels, measured by Northern blot analysis with a rat reg cDNA probe (n = 5), increased 300% at 2 days in the wrapped lobe and decreased to that of unwrapped controls by 14 days. In situ hybridization showed localization of reg to the acinar cells. In unwrapped gastric lobes of animals who underwent surgical wrapping of the splenic lobe, no change in islet number per square millimeter or reg gene expression was noted. CONCLUSIONS: Surgical wrapping of the pancreatic splenic lobe induces local reg gene expression that is temporally associated with duct cell hyperplasia. This is followed by islet formation within the wrapped lobe. Reg may play a role in the induction of new islets from ductular precursors and in maintenance of normal islet function.

Pancreatic thread protein is mitogenic to pancreatic-derived cells in culture.

BACKGROUND & AIMS: Pancreatic thread proteins (PTPs) are acinar cell products and members of the regenerating gene (reg) family. reg expression increases during islet regeneration, is depressed during aging-related islet dysfunction, and may be important in beta-cell growth and maintenance. The aim of this study was to examine the genetic expression of reg in pancreatic-derived cells in vitro and the mitogenic effect of PTP/reg protein on these cells. METHODS: reg gene expression was measured by Northern analysis in three rat pancreatic cell lines: ARIP (ductal), AR42J (acinar), and RIN (beta-cell). PTP/reg protein was isolated from bovine and human pancreas. Cells were cultured with PTP/reg for 72 hours, and thymidine incorporation was measured. RESULTS: reg messenger RNA was detected in AR42J but not in ARIP or RIN. PTP/reg protein was mitogenic to RIN and ARIP in a dose-related fashion but not to AR42J. It was not mitogenic to cultured mature rat islets. CONCLUSIONS: reg messenger RNA is expressed in acinar but not in beta-cell or ductal pancreatic cell lines. PTP/reg protein was mitogenic to both beta-cell and ductal cell lines but not to mature, nondividing islets. This supports the hypothesis that PTP/reg protein is an acinar cell-derived mediator of beta-cell growth and may be involved in modulating the duct-to-islet axis.