ABSTRACT
OBJECTIVE: To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent. METHODS: Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification. RESULTS: The titer of cDNA library was 3.85 × 10(7) pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20). CONCLUSIONS: The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.
Subject(s)
DNA, Protozoan/genetics , Gene Library , Giardia lamblia/genetics , DNA, Protozoan/chemistry , Giardia lamblia/chemistry , Giardiasis/parasitology , Humans , Polymerase Chain Reaction/methods , RNA/chemistry , RNA/genetics , RNA/isolation & purification , Trophozoites/chemistryABSTRACT
<p><b>BACKGROUND</b>The role of the cerebral venous system (CVS) in intracranial pressure (ICP) regulation remains largely unclear. In the present study, the interaction between ICP and the cerebral venous system and its possible mechanism were investigated with respect to the biological characteristics of the cerebral venous system and its hemodynamic response under increased ICP.</p><p><b>METHODS</b>We created intracranial hypertension animal model, measured and calculated the venous flow velocity and diameter of the outflow terminal of the CVS with color ultrasonic system and recorded the vascular morphology by 3-dimensional anatomical microscopy. Patients who suffered from raised ICP underwent MRI and digital subtraction angiography (DSA) examination to show the length in the vertical direction of the wall of the bridging vein representing the diameter value. Pathological autopsy was performed from bodies of patients who had died from non-cerebral causes to observe the juncture part between the venous sinuses and tributary vertical brain veins.</p><p><b>RESULTS</b>Under increased ICP conditions, venous drainage through the outlet cuff segment, a unique structure between the bridge vein and sinus, was obstructed and in turn venous blood became congested. Therefore, the increased blood volume worsened the pre-existing ICP according to the well-accepted theory regarding volume-pressure relationship. This phenomenon was described as concurrent "venogenic intracranial hypertension", which is characterized by intracranial venous blood stasis responsive to and together with the original increased ICP.</p><p><b>CONCLUSIONS</b>The existence of this special pathophysiological process is prevalent, rather than rare, in various intracranial disorders. This finding would definitely provide new insight into the area of cerebral venous system research.</p>
