Blood Sample Collection (Acr Criteria For Sle Patients)

The American College of Rheumatology (ACR) classification criteria were developed for clinical studies of lupus. These criteria were developed and validated for the classification of patients with a longstanding established disease and may exclude patients with early disease or disease limited to a few organs.
Sno. Criterion Definition
1. Malar Rash
Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds
2. Discoid rash
Erythematous raised patches with adherent keratotic scaling and follicular plugging; atrophic scarring may occur in older lesions
3. Photosensitivity
Skin rash as a result of unusual reaction to sunlight, by patient history or physician observation
4. Oral ulcers Oral or nasopharyngeal ulceration, usually painless, observed by physician
5. Nonerosive Arthritis Involving 2 or more peripheral joints, characterized by tenderness, swelling, or effusion
6. Pleuritis or
Pericarditis
1. Pleuritis--convincing history of pleuritic pain or rubbing heard by a physician or evidence
of pleural effusion OR
2. Pericarditis--documented by electrocardigram or rub or evidence of pericardial effusion
7. Renal Disorder 1. Persistent proteinuria > 0.5 grams per day or > than 3+ if quantitation not performed OR
2. Cellular casts--may be red cell, hemoglobin, granular, tubular, or mixed
8. Neurologic Disorder 1. Seizures--in the absence of offending drugs or known metabolic derangements; e.g., uremia, ketoacidosis, or electrolyte imbalance
OR
2. Psychosis--in the absence of offending drugs or known metabolic derangements, e.g., uremia, ketoacidosis, or electrolyte imbalance
9. Hematologic
Disorder
1. Hemolytic anemia--with reticulocytosis OR
2. Leukopenia--< 4,000/mm3 on ' 2 occasions OR
3. Lyphopenia--< 1,500/ mm3 on ' 2 occasions OR
4. Thrombocytopenia--<100,000/ mm in the absence of offending drugs
10. Immunologic
Disorder

1. Anti-DNA: antibody to native DNA in abnormal titer OR
2. Anti-Sm: presence of antibody to Sm nuclear antigen OR
3. Positive finding of antiphospholipid antibodies on:
' an abnormal serum level of IgG or IgM anticardiolipin antibodies,
' a positive test result for lupus anticoagulant using a standard method,
' a false-positive test result for at least 6 months confirmed by Treponema pallidum immobilization or fluorescent treponemal antibody absorption test
11. Positive Antinuclear
Antibody
An abnormal titer of antinuclear antibody by immunofluorescence or an equivalent assay at any point in time and in the absence of drugs

' When the patients are classified according to the ACR criteria for Lupus then Blood samples can be collected from them. Blood samples were collected in a heparnized 5ml disposable syringes.

Blood sample collection
' For the comparative study blood samples were also collected from the Healthy controls.
2.2 RNA Isolation
Reagents used for RNA isolation: RBC Lysis Buffer, Phosphate Buffer Saline, Trizol, Chloroform, Isopropenol, 70% Ethanol, DEPC Water.
Reagents for gel: 1%agrose gel, Gel running buffer, RNA loading dye
2.2.1 Reagent Preparation:
S.No Reagent Name Materials mM/%age used
1. DEPC water Di Ethyl Pyro Carbonate (DEPC)
Distilled Water 0.05%
2. RBC Lysis Buffer
pH: 7.4 Ammonium Chloride (NH4Cl)
Sodium Bicarbonate (NaHCO3)
EDTA 155mM
12mM
0.1mM (pH:0.5)
3. Phosphate Buffer Saline
pH: 7.4 Sodium Chloride (NaCl)
Potassium Chloride (KCl)
Na2HPO4
KH2PO4 137mM
2.7mM
10mM
2mM
4. Sodium Phosphate Buffer pH: 7.4 Na2HPO4
NaH2PO4 1000mM
1000mM
5. Gel running Buffer Sodium Phosphate Buffer
Formyldehyde
Volume make with DEPC water
6. RNA loading Dye Formamide
SDS
Bromophenol Blue
Ethidium Bromide
EDTA 95%
0.025%
0.025%
0.025%
0.5 mM

Important: All the reagents for RNA isolation are prepared in DEPC water. All the tips or tubes used in RNA isolation or its regent preparation must be DEPC treated.
2.2.2 DEPC Treatment
' 0.05% of DEPC water made in distilled water.
' All the tips and tubes to be used for RNA isolation were dipped in DEPC water .
' Leaving it dipped at 370C overnight.
' Next day autoclaving tips, tubes and DEPC water for 40 minutes at 15 lbs pressure.
' Allow the tips and tubes to dry properly in hot air oven by straining its DEPC water carefully.
' Once the tips are dried properly they are ready to use for the RNA isolation.
2.2.3 RNA isolation (for 1.5ml of blood)
' 1.5ml of fresh blood was taken in a falcon tube.
' Of its 4 times RBC Lysis buffer was added. (about 6ml)
' Mixed by inverting the tube slowly.
' Incubated for 10minutes at room temperature.
' Spin for 5 minutes at 2000 rpm for 10 minutes.
' Discard the supernatant and break the pellet.
' Add 5ml of Phosphate buffered Saline. Mix slowly by inverting.
' Centrifuge for 10minutes at 2000 rpm.
' Discard the supernatant and break the pellet.
' Add 1ml of Trizol, mix by vigorous pipetting and vortexing.
' Stand it for 5minutes. Transfer in an eppendorff tube.
' Add 0.2ml chloroform. Shake for 5 minutes (not vortexing).
' Stand it for 15 minutes at room temperature.
' Centrifuge at 12000g for 15 minutes at 40C.
' Take upper aqueous phase in fresh tube.
' Add 0.5ml of isopropenol. Mix gently by inverting the tube.
' Stand it for 10 minutes.
' Centrifuge at 12000g for 8 minutes at 40C
' For RNA precipitation remove the supernatant and wash pellet by adding 1ml of 75% ethanol (ice cold).
' Centrifuge at 12000g for 5 minutes at 40C.
' Remove ethanol, air dry the pellet (5 minutes).
' According to the pellet size dissolve the pellet in DEPC water (maximum in 30??l).
' RNA sample was taken into two aliquots
' For Quantification (2??l RNA sample + 8??l DEPC water)
' For running in agarose gel electrophoresis (3??l)
' Store the aliquots at -800C for further use.
2.2.4 Running an Agarose Gel Electrophoresis for checking the presence of RNA
1% agarose gel was prepared
' According to the volume Agarose was added into DEPC water and 0.1M Phosphate buffer.
' Agarose was allowed to boil in DEPC and phosphate buffer until the clear solution is obtained.
' Formaldehyde is added into the solution.
' Melted Agarose was poured into the caster and allowed to settle with comb into it.
' Once gel is settled properly into the caster the keep the gel into the Gel running tank with Running buffer into it. Now the combs were removed carefully.
Running buffer and RNA loading dye was prepared according to the composition given above.
RNA sample preparation for loading into the gel
' RNA loading dye (3??l) was added with one of the aliqout (3??l) of RNA sample.
' Now sample was kept at 60??C for 5-10 minutes (For denaturation).
' After 5-10 minutes immediately keep the sample on ice.
' Spin and load the sample on 1% Agarose gel.
' Allow the gel to run at 50volts.
Once the sample ran half of the gel the image was taken into the Gel Doc in UV light.

2.3 RNA quantification
Before proceeding for the DNAse treatment RNA quantification was done with Nanodrop to optimize the amount of other reactants to be added for DNase treatment or cDNA preparation.

Nano Drop machine
2.4 DNase treatment
DNAse treatment was done to remove other genomic (e.g degraded DNA) contamination present with our RNA sample. DNAse treatment was performed with DNAse treatment kit.
Kit composition
Sno. Components Kit concentration Conc. To be used
1 DNAse Buffer 10x 1x
2 DNAse I Enzyme 2U/??l 1U
3 RNAse Inhibitor 10U/??l 1U
4 DEPC water -- To make up the volume

' All the components were added in an eppendorf tube, final volume was make up with DEPC water.
' RNA samples were added according to their quantified concentration.
' Incubated for 15 minutes at 37??C.
2.5 DNAse I Enzyme Inactivation
DNAse inactivation is an important step; it is done so that cDNA to be prepared in further steps must not get degraded with DNAse I enzyme.
' 1??l(5mM) of 25mM concentration EDTA was added.
' Incubated at 65??C for 15 minutes to inactivate DNAse I enzyme.
' Incubated on ice for further use.
2.6 cDNA Preparation
cDNA preparation was done for studying regulation of our gene of interest by performing Real Time PCR with the cytokine specific primers. cDNA preparation was done by performing reverse transcriptase PCR which makes cDNA from RNA samples.
Reverse Transcriptase PCR was performed with cDNA preparation kit.
Kit Composition
SNo. Components Kit concentration Conc. To be used
1 Reverse Transcriptase Buffer 10x 1??l
2 Random Primer 10mM 1??l
3 Di Neucleotide triphosphate's mix 10mM 1??l
4 Reverse Transcriptase Enzyme 5U/??l 2??l
5 RNAse Inhibitor 10U/??l 1??l
6 DEPC miliQ water ------ To make up volume

' All the components were added in an eppendorf tube, final volume was make up with DEPC water.
' RNA samples were added according to their quantified concentration.
' Reverse Transcriptase PCR was allowed to run according to the number of cycles to be performed.

Reverse Transcriptase PCR machine
2.7 Real Time PCR with Housekeeping gene (e.g. Beta Actin)
Q-PCR with housekeeping was performed to check out that our cDNA has been prepared significantly or not. When getting good results with housekeeping gene then only one can proceed further for PCR with gene (cytokine) of our interest with its specific primers.
Q-PCR composition (concentrations are previously optimized)
S.No. Components Concentration
1 Dream Taq master mix (2x) 7.5??l
2 Forward Primer (for Beta Actin) 0.3??l
3 Reverse Primer (for Beta Actin) 0.3??l
4 MgCl2 0.9??l
5 MiliQ water 4.7??l

' All the components were added in a PCR tube, final volume make up with Mili Q water.
' 1.5??l cDNA templates of SLE patients and controls were added individually in each PCR tubes for which PCR is to be performed.
' Q-PCR was allowed to run according to the number of cycles of amplification.(35 cycles)

Real Time Thermo recycler

2.7.2 Running an Agarose Gel Electrophoresis for checking the amplified DNA (PCR product)
Sno Reagent Name Materials Concentration
1. 5X Tris Boric EDTA buffer
(For running the gel at 1x) Tris-borate
Boric acid
0.5 M EDTA, pH 8.0
Distilled water 54g
27.5g
20ml
To make up the volume upto 1L
2. DNA loading Dye Bromophenol blue
Glycerol
NaCl
Distilled water 0.26 g
30 ml
1 M
To make up the volume upto 100ml

2.5% Agarose Gel was prepared to load the PCR product
' 5x TBE buffer was diluted to 1x and agarose was added into it according to its volume.
' Heated to boiling until the clear solution was formed.
' When solution became Luke warm 55??C, then Ethidium Bromide was added to it.
' Melted Agarose was poured into the caster and allowed to settle with comb into it.
' Once gel is settled properly into the caster the keep the gel into the Gel running tank with Running buffer into it. Now the combs were removed carefully.
DNA Sample loading into the gel
' 3??l of 6x DNA loading dye was added to the PCR product.
' Samples were spined and mixed by pipetting.
' Now samples were loaded carefully into the wells.
' Gel was allowed to run at 100volts.
Once the sample ran half of the gel the image was taken into the Gel Doc in UV light.

2.8 Real Time PCR with the Specific primers of the Cytokine of interest IL-22
2.8.1 Primer Designing
Before performing the PCR with specific primer, one should know how get the specific Primer for a cytokine. Primers can be designed or can be chosen published ones. Here for Cytokine IL-22 Primers were designed through NCBI portal using PRIMER BLAST tool.

' NCBI portal was opened with url http://www.ncbi.nlm.nih.gov/
' Parameters was set according to our reaction.
' As soon as getting the primer exactly to parameters or near to it then the primers were ordered.

2.8.2 Primer dilution and stock preparation
As primers are packaged in lyophilized forms so when getting the primers they must be dissolved into the Mili Q water and stock should be prepared of 200 pico moles. These dissolved primers were left at least for 2-3 hrs at -20??C to mix thoroughly before starting with work.
Working concentration of primers are 10 pico moles so further dilution of primers are done with MiliQ water.
' Immediate stock (50pm)
' Working concentration (10pm)
2.8.3 Primers Optimization
Optimizations of primers are done to check the proper annealing temperature of the primer. This is done by running a gradient PCR with different temperatures. Temperatures to be optimized must be near to the Melting temperature of the primers. Once getting the appropriate temperature then RT-PCR can be performed with the patients sample and control samples.
2.8.4 Real time PCR with IL-22
As done with Housekeeping gene, all the components were similar except Primers.
Q-PCR composition (concentrations are previously optimized)
S.No. Components Concentration
1 Dream Taq master mix (2x) 7.5??l
2 Forward Primer (for IL-22) 0.3??l
3 Reverse Primer (for IL-22) 0.3??l
4 MgCl2 0.9??l
5 MiliQ water 4.7??l

' All the components were added in a PCR tube, final volume make up with Mili Q water.
' 1.5??l cDNA templates of SLE patients and controls were added individually in each PCR tubes for which PCR is to be performed.
' Q-PCR was allowed to run according to the number of cycles of amplification.(35 cycles)
' PCR products were allowed to run on 3% Agarose Gel as done with Housekeeping gene.
Comparative studies can be performed among the images of Housekeeping gene and IL-22 to study cytokines expression level.

Source: Essay UK - http://www.essay.uk.com/free-essays/science/blood-sample-collection.php


Not what you're looking for?

Search:


About this resource

This Science essay was submitted to us by a student in order to help you with your studies.


Rating:

Rating  
No ratings yet!

  • Order a custom essay
  • Download this page
  • Print this page
  • Search again

Word count:

This page has approximately words.


Share:


Cite:

If you use part of this page in your own work, you need to provide a citation, as follows:

Essay UK, Blood Sample Collection (Acr Criteria For Sle Patients). Available from: <https://www.essay.uk.com/free-essays/science/blood-sample-collection.php> [26-05-19].


More information:

If you are the original author of this content and no longer wish to have it published on our website then please click on the link below to request removal:


badges