Isolation RNA from whole blood by using ZR Whole-Blood RNA MiniPrep™

The ZR Whole-Blood RNA MiniPrep™ provides a streamlined method for the rapid isolation of total RNA from whole or partitioned blood in as little as 10 minutes.
RNA can be isolated immediately from fresh samples or at a later time from blood stored in Blood RNA Buffer.  This product is designed to isolate RNA from blood for subsequent RNA-based methods including RT-PCR, hybridization, etc.

The Flowchart

source: zymo

Epigenetic: RNA methylation by using EZ RNA Methylation Kit

What is Epigenetic?

Biological mechanisms in which DNA, RNA, and proteins are chemically or structurally modified, without changing their primary sequence (a.k.a mutation). 

Epigenetic regulatory mechanisms include DNA methylation and hydroxymethylation, histone modification, chromatin remodeling, RNA methylation, and regulation by small and long non-coding RNAs.

What is RNA methylation?

 Although the majority of nucleic acid modification research involves 5-methylcytosine (5-mC) in DNA, RNA is also extensively modified. In fact, there exist over a hundred modifications to RNA, including 5-mC, and RNA methylation is a common and naturally-occurring event in both prokaryotic and eukaryotic organisms. RNA methylation has been observed in numerous types of RNA molecules, including mRNAs, tRNAs, and non-coding RNAs, but the function of RNA methylation remains unclear.

Source: Zymo

How to use EZ RNA Methylation Kit ?

Follow the following flowchart and the complete protocol here

Source: Zymo

What happened to RNA methylation?

Sequencing results following bisulfite treatment. RNA with methylated C (5-mC) at nucleotide position #7 was processed using the EZ RNA Methylation™ Kit. The recovered RNA was amplified by RT-PCR and then cloned and sequenced. The methylated cytosine at position #7 remained intact while the non-methylated cytosines at positions #3, 4, and 13 were completely converted into uracil (post-bisulfite treatment) and detected as thymine following RT-PCR and sequencing

 

 Source: Zymo

Epigenetic: bisulfite treatment using Zymo kit (DNA methylation)

What is Epigenetic?

Biological mechanisms in which DNA, RNA, and proteins are chemically or structurally modified, without changing their primary sequence (a.k.a mutation). 

Epigenetic regulatory mechanisms include DNA methylation and hydroxymethylation, histone modification, chromatin remodeling, RNA methylation, and regulation by small and long non-coding RNAs.

Is it normal for our body to have DNA methylation?

 Yes, it is normal for our body, in fact recently many diseases due to aberration in epigenetic function. 

In complicated terms these epigenetic modifications normally play critical roles in the regulation of numerous cellular processes, including gene expression, DNA replication, and recombination

Source: Zymo

What kind of disease due to epigenetic aberration?  

 Well the most obvious is cancer.

Source: uiuc.edu

 

What is DNA methylation?

DNA which has methyl group (-CH3) on its nucleotide (especially Cytosine or C in mammals)

Source: uiuc.edu

Or in complicated term DNA methylation: Methylation of cytosine is a covalent modification of DNA, in which hydrogen H5 of cytosine is replaced by a methyl group 

Source: Sigma Aldrich

 

What is bisulfite treatment?

Converting C to be U by using sodium metabisulfite

What is bisulfite protocol?

What kind of kit to choose?

Zymo Research has several kit depend on your need, the following is recommended to see.

Source:
Zymo Research

Gomori Aldehide Fuschin Staining for Pancreatic Beta Cell and Elastic Fiber

Protocol After Paraffin Block Sectioning

1. Rehydration (Xylene 2x5 minutes, ethanol 100% 2 minutes, ethanol 95% 2x2 minutes, ethanol 70% 2 minutes, deionized water 2 minutes) 
2. Gomori Aldehide Fuschin Staining 1 hours then wash deionized water 10 dips
3. Nuclear Fast Red for 5 minutes then wash in tap water for 5 minutes  
4. Dehydration (ethanol 70% 10 seconds, ethanol 95% 20 seconds, ethanol 100% 10 seconds, xylene 2x10 seconds) 
5. Mounting in Xylene based medium

Result

Recepie for Gomori Aldehide Fuschin:

1. Weight 2.4 gr picric acid solid to a bronze bottle, added with water to 200ml, shaking for 5-10mins (Picric acid stored in 36% water, so pulled out water before weighting, remember to return back the water to the bottle after weighting)
2. Add 0.2 gr Direct Red 80 to above solution, then shaking/mixing for 5 minutes

Recepie for Nuclear Fast Red:

1. Weight 2.4 gr picric acid solid to a bronze bottle, added with water to 200ml, shaking for 5-10mins (Picric acid stored in 36% water, so pulled out water before weighting, remember to return back the water to the bottle after weighting)
2. Add 0.2 gr Direct Red 80 to above solution, then shaking/mixing for 5 minutes

Recommended Reagents:

1. Paraldehyde (Sigma Aldrich) 
2. 
   
3. Nuclear Fast Red (Sigma Aldrich)

Collagen Staining by Picro-Sirius Red

Protocol After Paraffin Block Sectioning

1. Rehydration (Xylene 2x5 minutes, ethanol 100% 2 minutes, ethanol 95% 2x2 minutes, ethanol 70% 2 minutes, deionized water 2 minutes) 
2. Picro-Sirius Red staining 1 hours then wash deionized water 10 dips
3. Gill Hematoxylin for 5 minutes then wash in tap water for 5 minutes  
4. Dehydration (ethanol 70% 10 seconds, ethanol 95% 20 seconds, ethanol 100% 10 seconds, xylene 2x10 seconds) 
5. Mounting in Xylene based medium

Result

Recepie for Picro-Sirius Red:

1. Weight 2.4 gr picric acid solid to a bronze bottle, added with water to 200ml, shaking for 5-10mins (Picric acid stored in 36% water, so pulled out water before weighting, remember to return back the water to the bottle after weighting)
2. Add 0.2 gr Direct Red 80 to above solution, then shaking/mixing for 5 minutes

Recommended Reagents:

1. Picric acid (Sigma Aldrich) NOTE: Becareful picric acid is toxic and potentially explosive when dry)
2. Direct Red 80 (Sigma Aldrich)

Detection of MMP1 by Immunohistochemistry in Cancer, Aging and Rheumatoid Arthritis

MMPs or matrix metalloproteinases are member of peptidase. These enzymes have capability for degradation extracellular matrix components (collagen, gelatin, Fibronectin, Laminin and proteoglycan. MMP genes can be activated by phorbol ester, lipopolysaccharide (LPS) or staphylococcal enterotoxin B (SEB). For doing its job, MMP requires both calcium and zinc. MMP-1 is downregulated by p53, and abnormality of p53 expression may contribute to joint degradation in rheumatoid arthritis by regulating MMP-1 expression.

  

IHC of MMP1 in Mouse Lung (Abbiotec)


Western blott Analysis of Human MMP1(Santa Cruz)

Principal of Protocol: 

1. Rehydration (Xylene 2x5 minutes, ethanol 100% 2 minutes, ethanol 95% 2x2 minutes, ethanol 70% 2 minutes, PBS 2 minutes) 
2. Antigen Retrieval (Citrate buffer in 800 Watt Microwave for 5 minutes) then wash PBS 2x5 minutes 
3. Blocking for endogenous peroxidase (Hydrogen Peroxide 3% for 5 minutes)then wash PBS 2x5 minutes 
4. Primary Antibody (1 hours in room temperature 25 degree Celcius)then PBS 2x5 minutes 
5. Secondary Antibody (Depend on manufacture, in this case using LSAB+ Kit Dako), then wash 2x5 minutes 
6. Dehydration (ethanol 70% 10 seconds, ethanol 95% 20 seconds, ethanol 100% 10 seconds, xylene 2x10 seconds) 
7. Mounting in Xylene based medium 

Recommended Reagent:

1. Citrate Buffer (Trisodium Citrate Sigma Aldrich) 
2. Primary Antibody (Santa Cruz, Abbiotec) 
3. Secondary antibody LSAB+ kit (Dako) 
4. Xylene or substitute (Merck, Shandon) 

Citation:
Santa Cruz
Abbiotec
DAKO

Sandwich ELISA

sandwich elisa process

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