In all cases samples were used with informed consent of subjects

In all cases samples were used with informed consent of subjects. The approval of the Institutional Ethics Committee was obtained for these studies (KHEC-31/2005). The cancer patients were at different stages of cancer of the cervix. All samples were collected from patients who came for treatment. This so has resulted in availability of very few samples from stages other than II and III, for example, CIN 1, CIS, and so forth, 19 patients were in stage III, 7 in stage II, 1 stage 0 (CIN I), 1 stage IV, and 1 from dysplasia of cervix. A total of 15 normal samples and 29 malignant samples were analyzed. All the malignant samples were of squamous cell carcinoma. The sample details are given in Table 1.Table 1Sample details.

All the samples, irrespective of whether they belonged to normal or cancer patients, were transported to the lab immediately after collection in normal saline. In the lab the tissues were washed with saline several times to remove any traces of blood. If the tissue samples were to be stored, they were immediately frozen in liquid nitrogen and stored at ?80��C in the deep freeze. They were passively thawed to room temperature just before use. We have verified that this procedure did not show any noticeable difference in the protein profile of a given sample. The samples were weighed and minced with 20% wet weight of Tris-EDTA buffer. They were then homogenized by a manual homogenizer (T8 blade IKA-WERKE), centrifuged at 5000rpm for 20 minutes twice. Supernatant was collected through a syringe fitted with 0.45micron filter.

50 microliters of the sample homogenate was injected into the HPLC-LIF system, which had a 20 microliter loop.2.3. Data AnalysisData processing of recorded protein profiles involved background correction, smoothing, calibration, and normalization [14]. All protein profiles were normalized Carfilzomib with respect to the 1594 seconds peak, which remained more or less constant in all samples. Data analysis was done by Principal Component Analysis (GRAMS/32, PLS PLUS/IQ software, in Galactic Corporation, USA). Diagnosis of tissue type as normal/malignant was achieved by classification of samples using Match/No Match condition of statistical parameters to those of normal and malignant calibration sets. The details of these have already been discussed in our earlier paper [14].To start with, PCA was run with all the samples, (15 normal and 29 malignant), combined, irrespective of whether they belong to normal or malignant group. The analysis was performed using 12 factors. PCA was extended further to see whether a given tissue sample can be identified more objectively as belonging to a specific group, say, normal or malignant.

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