An improved method for isolating Schwann cells from postnatal rat sciatic nerves

The major difficulty in Schwann cell (SC) purification is contamination by fibroblasts, which usually become the predominant cell type during SC enrichment in vitro. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. Our objectives have been to develop an efficient, easily applicable, rapid method to obtain highly purified SC from the sciatic nerve of newborn rats. The method involves two rounds of purification to eliminate fibroblasts with the novel combined use of cytosine-B-arabinoside hydrochloride (Ara-C) action and differential cell detachment. Cultured cells were first treated with Ara-C for 24 h. The medium was replaced with the growth medium containing 20 ng/ml human heregulin1-β1 extracellular domain (HRG1-β1 ECD). After another 48 h in culture, the cells were treated with 0.05% trypsin, following which SCs, but not fibroblasts, were easily detached from the dishes. The advantage of this method is that the two steps can eliminate the fibroblasts complementarily. Ara-C eliminates most of the fibroblasts growing among SCs, whereas the differential cell detachment technique removes the remainder growing under or interacting with the SC layer. A purity of more than 99% SCs has been obtained, as confirmed by cell morphology and immunostaining. The purified SCs have a spindle-shaped, bipolar, and sometimes tripolar morphology, align in fascicles, and express S-100. The whole procedure takes about 10 days from primary culture to the purified SCs growing to confluence (only half the time reported previously). This protocol provides an alternative method for investigating peripheral nerve regeneration and potentially could be used to produce enough SCs to construct artificial nerve scaffolds in vitro. This work was supported by Tsinghua-Yue-Yuen Medical Sciences Fund, the National Natural Science Foundation of China (contract grant numbers: 30670528, 30700848, 30772443), Beijing Municipal Science & Technology Commission (BMSTC, contract grant number: H060920050430), National Basic Research Program of China (also called the 973 Program, contract grant number: 2005CB623905), and the National Natural Science Foundation of Beijing (contract grant number: 7082090).     

An improved method for isolation of RNA from bone

Background  

Bone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes. However, progress in understanding the role of bone in determining metabolic health is hampered by the well-described difficulty of obtaining high quality RNA from bone for gene expression analysis using the currently available approaches.     

An improved method of isolating Pneumocystis carinii from infected rat lungs

A new method of isolating Pneumocystis carinii from infected lungs of cortisonized rats is described. Clumping of parasites and host lung material was diminished by suspension of macerated Pneumocystis-laden rat lung in a modified calcium, magnesium-free Hanks' balanced salts solution at physiologic pH and osmolality, containing the wetting agent G-acid. After washing, this material was suspended in a second buffer system for digestion. The digestion step was done in the same buffer but with the addition of calcium, magnesium, collagenase, hyaluronidase and deoxyribonuclease. These innovations allowed enumeration of trophozoites as well as cysts. Following digestion, the parasites were separated from particulate host lung debris by Percoll density gradients designed to pellet the debris, leaving parasites in the gradient. Density studies done prior to this step revealed that trophozoites and non-nucleated cysts had similar densities, 1.028 g/ml, whereas nucleated cysts were heavier at 1.030 g/ml. Particulate host lung debris could be removed due to its heavier density, 1.040 g/ml. The significance of this study includes: relatively clump-free suspensions of infected rat lung, enumeration of trophozoites as well as cysts, and characterization of nucleated cysts.     

An improved method for isolating chloroplasts retaining their outer membranes

A new method of obtaining chloroplasts retaining their outer membranes is described. It is shown that the use of low cation media enhances the separation of intact and broken chloroplasts.     

A method for isolating total RNA from pear leaves

Isolation of high quality RNA fromRosaceae species is particularly difficult. These plants contain considerable amounts of plant polyphenolic compounds and polysaccharides that copurify with RNA, often rendering it unsuitable for either cDNA synthesis and/or hybridization in northern analyses. We describe a method for RNA isolation from pear leaves that is modified from that of Manning (1990). The procedure includes i) an extraction with phenol and PVPP, to remove proteins and polyphenols ii) two purifications by LiCl, with a 2-butoxyethanol treatment between the LiCl steps. The method results in high quality RNA suitable for RT-PCR and northern blot experiments.     

An improved method for isolation of RNA from rat femur

Background: RNA isolation from ossified bone is a difficult and time-consuming process which often results in poor recovery of RNA. The yield is limited and might not be suitable for gene quantification studies by real time PCR. Methodology: The present study demonstrates RNA extraction from rat femur utilizing the silica column along with the trizol reagent. Quality of RNA was assessed by agarose gel analysis and its suitability for real-time PCR analysis was determined by β-actin Ct values. Results: The RNA isolated using silica columns in conjugation with trizol reagent resulted in higher yield of RNA and purity (A260/280=2.04; yield =1545.73 µg/ml) compared to the trizol method alone (A260/280=1.85; yield =571.2 µg/ml). Ct value of β actin obtained from RNA isolated by trizol method was higher than the Ct value obtained by trizol in conjugation with the column method (31.41 and 15.41 respectively). Conclusion: Combination of trizol along with silica column resulted in better quality and improved yield of RNA suitable for gene quantification by Real time PCR.     

黄檗（Phellodendron amuranse）叶片总RNA提取方法研究

以黄檗(Phellodendron amuranse Rupr.)叶片为材料,分别利用改进的盐酸胍法、Trizol法、CTAB法提取黄檗叶片总RNA,通过RNA产率、纯度、电泳图谱等分析,确立了1种从黄檗叶片中快速分离总RNA的方法。研究结果表明,改进盐酸胍法所提取的总RNA的A₂₆₀/A₂₈₀为1.928,28S和18S条带清晰谱图完整性好,而且具有产率高、时间短、成本低的特点,所提取的总RNA适用于mRNA分离、cDNA文库的建立、Northern杂交等分析。     

黄檗(Phellodendron amuranse)叶片总RNA提取方法研究

以黄檗(Phellodendron amuranseRupr.)叶片为材料,分别利用改进的盐酸胍法、Trizol法、CTAB法提取黄檗叶片总RNA,通过RNA产率、纯度、电泳图谱等分析,确立了1种从黄檗叶片中快速分离总RNA的方法。研究结果表明,改进盐酸胍法所提取的总RNA的A260/A280为1.928,28S和18S条带清晰谱图完整性好,而且具有产率高、时间短、成本低的特点,所提取的总RNA适用于mRNA分离、cDNA文库的建立、Northern杂交等分析。     

A micromethod for the isolation of total RNA from adipose tissue.

We have developed a simple and rapid procedure for the isolation of total RNA from small amounts of adipose tissue. Using this method, it is possible to obtain quantitative recovery of RNA from less than 300 mg of adipose tissue, with an average yield of 70 micrograms of RNA per gram of adipose tissue. Northern blot analysis of rat epididymal adipose tissue RNA samples was performed using a beta-actin probe and demonstrated that intact total RNA had been isolated. The procedure has been adapted for use in 1.5-ml microcentrifuge Eppendorf tubes, providing a convenient and inexpensive method for the reproducible recovery of intact RNA from sparse samples of adipose tissue.     

An improved method for isolating a section of the rat neocortex]

Neocortical island chronically isolated from surrounding cortical and subcortical structures with preserved pial blood supply has long been a model for research into the mechanisms of cortex functioning. To fully cut the cortex we improved the type of knife by using a retractable tungsten wire. The tip of a syringe needle was bent and cut away all but the beginning of the bend. In anesthetized rats the somatosensory cortex was exposed, the guide needle was lowered down to the desired depth into the cortex avoiding blood vessels. The wire then was pulled out through the curved needle tip until the tip of the wire touched the pia mater. The device was then raised, lowered, rotated to achieve complete separation of the cortical island from the surrounding tissues. The wire was retracted into the needle before withdrawal of the device. Analysis of neocortical slices 8 weeks later showed lesions of the white matter and transcortical cuts.     

An improved method for embedding brain tissue in albumin-gelatin.

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose, and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 micron. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.     

A simple and effective method for isolating RNA from alfalfa pollen

Extraction of RNA from alfalfa pollen using conventional grinding devices resulted in low yields of degraded RNA; degradation appeared to be related to the length of time required to break open the pollen grains with such devices. A glass syringe was modified to provide the restricted, shearing environment necessary for rapidly rupturing this type of tissue. This apparatus would be useful for extracting a variety of molecules from pollen of any species, but is particularly valuable for species which produce low amounts of relatively small pollen grains.     

An improved method for the isolation of total RNA from spurce tissues

After many unsuccessful attempts to obtain biologically active mRNAs from spruce (Picea abies) tissues using available protocols, we have adapted a procedure for the isolation of RNAs from needles, shoots, and callus ofPicea species. Our modifications permit the recovery of and an average of 300 μg RNA per g of needles that is suitable for translationin vitro, northern hybridizations, and the construction of cDNA libraries.     

An improved method for extraction of high-quality total RNA from oil seeds

Biotechnology Letters - Seeds of oilseed plants that contain large amounts of oil, polysaccharides, proteins and polyphenols are not amenable to conventional RNA isolation protocols. The presence...     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.