Author: Promega

Exploring the Land of the Silver Fern: South Island of New Zealand—Part I

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In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue is Part I of the adventures of Sarah Theos, a client support consultant, who used her award to travel to New Zealand.

Introduction

We went to New Zealand the first two weeks in December and were surprised that there was hardly anyone there. We had long stretches of the road entirely to ourselves and felt like we were the only ones around on many of the hikes. It was a magical feeling to take in the gorgeous scenery without another soul for miles. We, of course, purchased a book that told us about all of the “off the beaten path” hikes and sights that we must do and tried to check them all off the list! The locals were also extremely friendly and eager to chat. If we did come across any locals on the trails, they always stopped to talk and were eager to hear about where we were from and how we like the island so far. It was the beginning of summer so the days were long with the sun rising before 6am and setting around 10pm. We had plenty of daylight hours to explore.

Day 1: Christchurch

Starting in Washington, DC, we traveled over 29 hours door to door to get to Christchurch, NZ. Since we arrived around 7am, we decided to fight off the jetlag and go out to explore the city. I wasn’t quite sure what to expect in Christchurch, knowing that it suffered two devastating back to back earthquakes in 2010 and 2011. My first impression was that there were a lot of gravel parking lots everywhere and boarded up high rises. It looked like a ghost town. We checked into our hotel (built after the earthquakes) and took a walk down to the City Centre. The once beautiful cathedral sat in ruins, surrounded by large buildings in various states of decay. It was a sad sight to see. We then walked further down the street to the botanical gardens. The gardens are beautiful and we spent quite a while admiring the stunning Rose Garden. After exploring the gardens, we decided to drive up to the gondola to explore the city from a different angle.

The gondola takes you up to the top of Port Hills, about 500 meters above sea level. At the top you can see a 360 degree view from the Pacific Ocean and Christchurch to the Southern Alps and the surrounding Canterbury plains. We hiked to the top of Cavendish Bluff to take in the beauty. After the gondola ride, we decided to drive a little ways up the Banks Peninsula to Governor’s Bay where we found a cute little pub. Exhaustion set in around 5pm so we decided to head to a grocery store to stock up on snacks and water in preparation for our drive to the wild, west coast the next day. We passed out around 8pm. Continue reading “Exploring the Land of the Silver Fern: South Island of New Zealand—Part I”

Thank a Tech or Assistant

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Today’s #FridayFeeling is one of gratitude for all of those people who do the things that make our lives easier: lab techs, work-study students, undergraduate assistants. They put up with our requests and changes of mind and help keep our laboratory glassware clean, solutions sterile and experiments running. Do you have someone who helps you keep your experiments up and running?eh26

Inflammasomes and Pyroptosis

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In today’s post, guest blogger,  Martha O’Brien, PhD, provides a preview of her upcoming AAI poster and block symposium talk on the inflammasome, caspase-1 activity and pyroptosis.

Schematic of the Caspase-Glo 1 Inflammasome Assay.
Schematic of the Caspase-Glo 1 Inflammasome Assay.

Responding rapidly to microbial pathogens and damage-associated molecular markers is critical to our innate immune system. Caspase-1 is pivotal in this process leading to processing and release of essential cytokines and an immunogenic form of cell death, termed pyroptosis. Upon sensing pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), innate immune cells form inflammasome protein complexes that recruit and activate caspase-1 (canonical inflammasomes). In addition, other inflammatory caspases, 4 and 5 in humans and 11 in mice, directly bind bacterial lipopolysaccharides (LPS), triggering pyroptosis (non-canonical inflammasome). LPS-triggered non-canonical inflammasomes in mice and humans ultimately lead to canonical inflammasome engagement and caspase-1 activation (1–3).  Caspase-1 was originally termed interleukin converting enzyme (ICE) for its well-established role in processing IL-1ß and IL-18, two important inflammation cytokines. How caspase-1 mediates pyroptosis is less well understood, but is beginning to be delineated. Recently, a substrate of the inflammatory caspases, gasdermin D, was identified and its processed fragment, gasdermin-N domain, was shown to be required for pyroptosis in non-canonical inflammasome circumstances (4, 5). The precise role of gasdermin D in canonical inflammasome-triggered pyroptosis is still under investigation. Linking inflammatory caspases directly to pyroptosis is a notable step in understanding the mechanism of this important form of cell death.

Pyroptosis is clearly one means of releasing processed IL-1ß and IL-18 from the cell. However depending on the cell type and stimulus, there is evidence for inflammasome engagement, caspase-1 activation, and release of IL-1ß in the absence of cell death (6, 7). On the flip-side there is also evidence for caspase-1 mediated pyroptosis that helps clear bacteria, independent of IL-1ß and IL-18 involvement (8). To enable further studies on the inflammasome and in particular, assessing the connections between caspase-1 activation, pyroptosis, and cytokine release, Promega developed a new tool to conveniently monitor caspase-1 activation, the Caspase-Glo® 1 Inflammasome Assay.  This bioluminescent, plate-based assay is used to measure caspase-1 activity directly in cell cultures or to monitor released caspase-1 activity in culture medium from treated cells. This flexibility allows easy multiplexing to monitor all three outcomes of inflammasome stimulation; caspase-1 activity, pyroptosis, and release of IL-1ß and IL-18. Caspase-1 activation typically is monitored indirectly with western blots of processed caspase-1. Now the activity of the enzyme can be monitored directly, providing accurate information on temporal aspects of the inflammasome. The assay can be readily combined with real-time measures of cell death (e.g., CellTox™ Green Cytotoxicity Assay) and some of the culture medium can be removed for IL-1ß/IL-18 assessment, leaving the cells and remaining culture medium for caspase-1 activity measurements.

References

  1. Schmid-Burgk et al. (2015) Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells. J. Immunol. 45, 2911–7.
  2. Baker et al. (2015) NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. J. Immunol. 45, 2918–26.
  3. Ruhl, S. and P. Broz (2015) Caspase-11 activates a canonical NLRP3 inflammasome by promoting K+ Eur. J. Immunol. 45, 2927–36.
  4. Shi et al. (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–5.
  5. Kayagaki et al. (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature 526, 666–71.
  6. Gaidt et al. (2016) Human monocytes engage an alternative inflammasome pathway. Immunity 44, 833–46.
  7. Chen et al. (2014) The neutrophil NLRC4 inflammasome selectively promotes IL-1ß maturation without pyroptosis during acute. Salmonella Cell Reports 8, 570–82.
  8. Miao et al. (2010) Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nature Immunology 11, 1136–42.

Helix® Personified

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Have you ever thought about life from the point of view of a Helix® On-Site Smart Inventory Unit? We did, and this is what we imagined…

helixtouchsceenisolatedWow! There were a lot of people in and out of here today.

The post doc who never goes anywhere without her earbuds in. I wonder what her research playlist is, she’s always dancing her way in and out of the hall. The new grad student in the virology lab next door. He’s looking a little lost right now.

And of course there’s the PI with the big boots. Never looks where he’s going and constantly almost plowing people down. It’s kind of funny actually, the near misses I’ve seen between him and grad students running out of here with that key reagent they need to finish those last few reaction tubes.

There was a big-name speaker today for the lunch time journal club, so there was a lot of activity on the hall this afternoon. Lots of talking about the presentation. Everyone sounded pretty excited about what they heard.

But now it’s quiet. Most people have gone home for the evening.

Except for the security lighting, the hall is dark.

Oh wait, I hear footsteps heading my way. Apparently, not everyone has stopped work for the day, apparently.

Oh, it’s you. One of my favorite after-hour scientists.

You open the door to the common room, and check the product list on your phone: 100bp DNA ladder … there’s one left. Drawer D.

You pull the pass out of your pocket. I scan it. Click. The door unlocks. You grab your package and let the door close.

I’ll take it from here so that you can get back to work.

Your pass tells me that you’re a post doc in a lab upstairs. After you close the door, I check to see what you took. My scan reads the tag on each reagent. G2101: 100bp DNA ladder. Drawer D.

That was the last one. I update the inventory list. More will arrive in the next resupply shipment.

I send you an email with the details.

Time to check my systems.

Power? Check.
Connection to Promega? Check.
Door closed and locked? Check.
Storage temperature holding steady? Check.
All reagents present? Check.

The lights go dark as I transmit the report back home.

There and Back Again: Part IV (South Island, New Zealand)

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In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue is Part IV and the final installment of the adventures of Mica Zaragoza, a senior client rep in Chicago, IL, who used his award to travel to Australia and New Zealand.

26061846-Helix-Travel---Image-1-WEBQueenstown, New Zealand 

Walking out of our room into the open air, Queenstown instantly provided a lasting impression.  Nestled along Lake Wakatipu, watched over by the Remarkable Mountains, the city feels like Aspen on steroids—it’s no wonder there’s such an international pull for young travelers and skiers.

We jumped right into exploring with a gondola ride. This photo is my best attempt at capturing Queenstown from high above. Continue reading “There and Back Again: Part IV (South Island, New Zealand)”

There and Back Again 3: Christchurch, New Zealand (South Island) Travelogue

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In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of the our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue is Part III of the adventures of Mica Zaragoza, a senior client rep, who used his award to travel to Australia and New Zealand.

Helix-Travel-Blog---1-WEBAfter a day of travel, including a quick stop-off in Sydney, we arrived late in New Zealand the evening.  New Zealand is an island nation with strict customs requirements, having been stung in the past by a decision  to import the opossum.

Jumping full into the experience, we rented a car (5-speed) and headed into the City to scope out our hotel ( more on the car later).

During our time in Australia, we had spoken with several travelers who had visited Christchurch for their recommendations.  Without fail, their response was negative, using words like “destroyed” and “rubble” to describe the city.  They referred to a  series of earthquakes in 2011 that crippled the city of Christchurch; the residents were not aware of the risk and structures in the city had not been constructed to withstand such devastation. Continue reading “There and Back Again 3: Christchurch, New Zealand (South Island) Travelogue”

There and Back Again: Part II (Cairns, Australia)

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In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of the our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue is Part II of the adventures of Mica Zaragoza, a senior client rep, who used his award to travel to Australia and New Zealand.

Cairns, Australia

24254847-Aussie-Kiwi-1Jumping from down South to way up North, the city of Cairns seems like a touristy beach town, sporting a laid-back feel with lots of cafes, pubs, and restaurants.  The locals drop the hard ‘R’s in most words, making the name of the city hit the ear more like “cans”.  Popping into town, we stopped by the city center and soaking pools along the Esplanade to relax.

With the Esplanade pools right next to the shoreline, I asked one of the locals why no one went swimming in the ocean. The answer was simple: underwater mangroves, mudflats, and saltwater crocs… more on that later. Continue reading “There and Back Again: Part II (Cairns, Australia)”

Real-Time (quantitative) qPCR for Quantitating Library Prep before NGS

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Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.
Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.

This the last in a series of four blogs about Quantitation for NGS is written by guest blogger Adam Blatter, Product Specialist in Integrated Solutions at Promega.

When it comes to nucleic acid quantitation, real-time or quantitative (qPCR) is considered the gold standard because of its unmatched performance in senstivity, specificity and accuracy. qPCR relies on thermal cycling, consisting of repeated cycles of heating an cooling for DNA melting and enzyamtic replication. Detection instrumentation is capable of measuring the accumulation of DNA product after each round of amplification in real time.

Because PCR amplifies specific regions of DNA, the method is highly sensitive, specific to DNA, and it can determine whether a sample is truly able to be amplified. Degraded DNA or free nucleotides, which might otherwise skew your quantiation, will not contribute to the signal, and your measurement will be more accurate.

However, while qPCR does provide technical advantages, the method requires special instrumentation, specialized reagents and is a more time-consuming process. In addition, you will probably need to optimize your qPCR assay for each of your targets to achieve your desired results.

Because of the added complexity and cost, qPCR is a technique suited for post-library quantitation when you need to know the exact amount of amplifiable, adapter-ligated DNA.  PCR is the only method capable of specifically targeting these library constructs over other DNA that may be present. This specificity is important because accurate normalization is especially critical for producing even coverage in multiplex experiments where equimolar amounts of several libraries are added to a pooled sample. This normalization process is essential  if your are screening for rare variants that might be lost in background and go undetected if underrepresented in a mixed pool.

 

Read Part 1: When Every Step Counts: Quantitation for NGS

Read Part 2: Nucleic Acid Quantitation by UV Absorbance: Not for NGS

Read Part 3: Fluorescence Dye-Based Quantitation: Sensitive and Specific for NGS Applications

ISO 18385: The Creation of a “Forensic Grade” Standard

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Today’s blog is from guest blogger Charles Stollberg. Charlie is a Promega production scientist in the Genetic Identity group and is focused on manufacturing inventory material and production process improvements. He’s been with the company for about 4 years. He graduated from UW-Whitewater in 2007 with a bachelor’s degree in cell biology. Prior to Promega, he worked in a small genetics lab studying lethal recessive traits in cattle.

ForensicGradeLogoForensic DNA laboratories rely on reagent and plastics manufacturers to supply high-quality products with minimal interference from contaminating DNA. With the increasing sensitivity of short tandem repeat (STR) amplification systems, levels of DNA that were previously undetected may now generate partial profiles. To address the concern of laboratories worldwide regarding the potential of low-level DNA contamination in consumables, ISO 18385 was developed to provide requirements for minimizing the risk of human DNA contamination events during the manufacturing process. Many of you may not have heard of ISO 18385, so I’d like to give you an introduction to how the standard came to be.

Continue reading “ISO 18385: The Creation of a “Forensic Grade” Standard”

Fluorescence Dye-Based Quantitation: Sensitive and Specific for NGS Applications

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This is the third post in a series of blogs on quantitation for NGS applications written by guest blogger Adam Blatter, Product Specialist in Integrated Solutions at Promega.

Fluorescent dye-based quantitation uses specially designed DNA binding compounds that intercalate only with double stranded DNA molecules. When excited by a specific wavelength of light, only dye in the DNA-bound state will fluoresce. These aspects of the technique contribute to low background signal, and therefore the ability to accurately and specifically detect very low quantities of DNA in solution, even the nanogram quantities used in NGS applications.

For commercial NGS systems, such as the Nextera Rapid Capture Enrichment Protocol by Illumina, this specificity and sensitivity of quantitation are critical. The Nextera protocol is optimized for 50ng of total genomic DNA. A higher mass input of genomic DNA can result in incomplete tagmentation, and larger insert sizes, which can adversely affect enrichment. A lower mass input of genomic DNA or low-quality DNA can generate smaller than expected inserts, which can be lost during subsequent cleanup steps, giving lower diversity of inserts.

Continue reading “Fluorescence Dye-Based Quantitation: Sensitive and Specific for NGS Applications”